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Keywords:

  • vascular invasion;
  • Cbfa1;
  • chondrocyte;
  • cartilage;
  • bone marrow

Abstract

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

Chondrocyte maturation and vascular invasion of cartilage are essential in the process of endochondral ossification. Cbfa1-deficient (Cbfa1−/−) mice displayed a complete absence of osteoblast and osteoclast maturation as well as severely inhibited chondrocyte maturation in most parts of the skeleton. Although chondrocyte maturation and mineralization were observed in restricted areas of Cbfa1−/− mouse skeleton, vascular invasion of calcified cartilage was never noted. To investigate the possibility of chondrocyte maturation and vascular invasion in Cbfa1−/− cartilage and the role of the hematopoietic system in the process of vascular invasion, we transplanted embryonic day 18.5 (E18.5) Cbfa1−/− femurs, which are composed of immature chondrocytes, into spleens of normal mice. One week later, the transplanted femurs contained terminally differentiated chondrocytes expressing osteopontin, bone sialoprotein (BSP), and matrix metalloproteinase (MMP) 13. In the diaphyses of the transplants, the cartilage matrix was mineralized and the cartilage was invaded by vascular vessels and osteoclasts. However, chondrocyte maturation and vascular invasion were severely retarded in comparison with transplants of E14.5 wild-type femurs, in which the cartilage was rapidly replaced by bone, and neither mature osteoblasts nor bone formation were observed. In primary culture of Cbfa1−/− chondrocytes, transforming growth factor (TGF) β1, platelet-derived growth factor (PDGF), interleukin (IL)-1β, and thyroid hormone (T3) induced osteopontin and MMP-13 expression. These findings indicated that factors in the hematopoietic system are able to support vascular invasion of cartilage independent of Cbfa1 but are less effective without it, suggesting that Cbfa1 functions in cooperation with factors from bone marrow in the process of growth plate vascularization.


INTRODUCTION

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

ENDOCHONDRAL OSSIFICATION is accomplished by sequential processes that include chondrocyte maturation, vascular invasion of cartilage, and bone formation by osteoblasts.(1) Cartilage initially is an avascular tissue, and chondrocyte maturation is a prerequisite for vascular invasion to occur in cartilage.(2) After chondrocyte maturation, a variety of angiogenic factors, including transferrin, vascular endothelial growth factor (VEGF), and matrix metalloproteinase (MMP) 13, are induced, and angiogenesis inhibitors, including chondromodulin I and tissue inhibitors of metalloproteinases (TIMPs), are suppressed.(3–7) Further, MMP-9, which is expressed mainly in osteoclasts, plays an important role in growth plate angiogenesis.(8) After vascular invasion has taken place, factors from endothelial cells continue to support chondrocyte maturation.(9)

Core binding factor α1/runt-related gene 2 (Cbfa1/Runx2) is a transcription factor that belongs to the runt-domain gene family.(10) Cbfa1−/− mice showed a complete lack of bone formation, including both intramembranous and endochondral ossification, showing that Cbfa1 is essential for osteoblast differentiation.(11,12) Further, Cbfa1 enhanced osteoblast differentiation at an early stage but inhibited it at a later stage of differentiation.(13) Cbfa1 is expressed in both osteoblasts and chondrocytes, and terminally differentiated chondrocytes (terminal hypertrophic chondrocytes) express abundant Cbfa1.(14,15) The maturation of chondrocytes in Cbfa1−/− mice was retarded, and the chondrocytes in most of the skeleton were resting or proliferating.(14,15) However, in restricted parts of the skeleton of Cbfa1−/− mice, including tibia, fibula, radius, and ulna, chondrocytes matured into hypertrophic chondrocytes and the cartilage matrix was mineralized. In terminal hypertrophic chondrocytes of the mutant mice, osteopontin, bone sialoprotein (BSP), and MMP-13, which are expressed normally in osteoblasts as well as terminal hypertrophic chondrocytes, were barely detectable.(14) Further, no mature osteoclasts were present, and vascular invasion into the cartilage was never noted, even in the calcified cartilage.(11,16) Although we and others have shown that Cbfa1 is required for chondrocyte maturation,(17–19) the reason for the complete lack of vascular invasion of the calcified cartilage of the mutant mice remains to be clarified.

Transplantation of cartilage into the spleen is a good model by which to examine vascular invasion into cartilage, because osteoclasts and blood vessels are supplied by the host.(20) Meckel's cartilage transplanted into spleen was invaded by blood vessels, and calcified matrix was generated by osteoblasts. Because the spleen is a hematopoietic organ in mice, cartilage transplanted into the spleen is able to acquire not only a sufficient amount of hormones but also factors released by hematopoietic cells and endothelial cells, such that cartilage acquires these factors from bone marrow in the process of endochondral ossification. Therefore, by transplanting cartilage into spleen it is possible to examine the role of the hematopoietic organ in chondrocyte maturation, vascular invasion, and matrix production by terminal hypertrophic chondrocytes that normally are in close contact with bone marrow.

Because Cbfa1−/− mice died just after birth,(11,12) it was impossible to investigate further chondrocyte maturation and vascular invasion of cartilage. Furthermore, Cbfa1−/− cartilage, which is resistant to vascular invasion, is a good material with which to evaluate the contribution of the hematopoietic system to the process of vascular invasion. To investigate the possibility of chondrocyte maturation and vascular invasion of Cbfa1−/− cartilage and the role of the hematopoietic organ in the process of vascular invasion, we transplanted embryonic day 18.5 (E18.5) Cbfa1−/− femurs, which consist mostly of immature chondrocytes, and E14.5 wild-type femurs into spleens of normal mice. Although we did observe chondrocyte maturation and vascular invasion of cartilage, these processes were severely retarded in Cbfa1−/− transplants, indicating that the hematopoietic organ is able to support the process of vascular invasion of cartilage but that this process is impaired in the absence of Cbfa1.

MATERIALS AND METHODS

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

Mice

Mice heterozygously mutated in the Cbfa1 locus (Cbfa1+/− mice) were generated as previously described.(11) Cbfa1+/− mice were backcrossed with C57BL/6 10 times and used for the transplantation experiments. Femurs and tibias were taken from Cbfa1−/− embryos at E18.5 and wild-type embryos were taken at E14.5. Muscle and connective tissues surrounding the perichondrium were removed under microscopic observation, and the femurs and tibias were used for transplantation into the spleen. Before the study, all experiments were reviewed and approved by Osaka University Medical School's committee on animal care and use.

Transplantation into the spleen

Four- to 5-month-old wild-type littermates of Cbfa1+/− mice were used as recipients for Cbfa1−/− and wild-type grafts. The recipient mice were anesthetized, and the grafts were transplanted carefully into the exposed spleens using a Venule-V-2 needle (Top Co., Ltd., Tokyo, Japan) as previously described.(20)

Histological and immunohistochemical examinations

For light microscopy, lower limbs from E18.5 Cbfa1−/− embryos and E14.5 wild-type embryos and spleens with the transplants were fixed at 3, 7, 14, and 21 days after transplantation in 4% paraformaldehyde/0.1 M phosphate buffer. Sections (3 μm in thickness) were stained with hematoxylin and eosin (H&E), von Kossa's stain, and TRAP.(11) For immunohistochemical analyses, endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide, and specimens were incubated with PBS containing 2% goat serum for 30 minutes at room temperature and then with monoclonal anti-VEGF antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or rabbit polyclonal antivacuolar H+-ATPase antibody (the kind gift of Y. Moriyama, Okayama University, Japan)(21) at 4°C overnight. The localization of the first antibodies was visualized by incubation with biotinylated goat anti-mouse immunoglobulin G2a (IgG2a; Santa Cruz Biotechnology Inc.) in VEGF and with biotinylated F(ab′)2 fragment of swine anti-rabbit immunoglobulins (DAKO, Glostrup, Denmark) in vacuolar H+-ATPase for 30 minutes at room temperature and then with avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. As negative controls, specimens were treated with second antibodies without incubation with first antibodies. The sections were counterstained with hematoxylin.

In situ hybridization, Northern blot, and reverse-transcription polymerase chain reaction

Probes for in situ hybridization and Northern blot analysis were as follows: a 0.4-kb fragment of mouse α1(II) collagen cDNA,(22) a 0. 5-kb fragment of rat α1(X) collagen cDNA,(23) a 1.2-kb fragment of mouse osteopontin cDNA,(24) a 0.7-kb fragment of mouse MMP-13 cDNA,(25) a 0.32-kb fragment of mouse α1(I) collagen cDNA,(22) and a 0.47-kb fragment of mouse osteocalcin cDNA.(26) A 0.54-kb (76-614 bp) fragment of rat BSP cDNA and a 0.85-kb (310-1164 bp) fragment of mouse GAPDH cDNA were amplified by polymerase chain reaction (PCR), subcloned into pBluescript (Stratagene, La Jolla, CA, USA), and checked by sequencing.

Digoxigenin-11-uridine triphosphate (DIG-11-UTP)-labeled single-stranded RNA antisense and sense probes for in situ hybridization were prepared using a DIG RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Hybridization was carried out as described.(11)

For Northern blot analyses, total RNA was extracted by lithium chloride from skeletons of E18.5 Cbfa1−/− and E14.5 wild-type embryos, transplants, spleen of wild-type mice, and cultured Cbfa1−/− chondrocytes.(27) Ten micrograms of total RNA was denatured with formamide, subjected to electrophoresis on 1.2% agarose gels, and transferred to nylon membranes. Membranes were hybridized with [32P]-labeled osteopontin, BSP, MMP-13, and GAPDH probes as previously described.(14)

For reverse-transcription (RT)-PCR analyses, the total RNA was reverse transcribed and amplified by Amp Taq DNA polymerase (Perkin Elmer, Norwalk, CT, USA) using the following primers: RANKL, 5′-GTCACTCTGTCCTCTTGGTAC-3′ and 5′-TGAAAGCCCCAAAGTACGTCG-3′; osteoprotegerin (OPG), 5′-CAGCTTCTTGCCTTGATGGAGA-3′ and 5′-AAACAGCCCAGTGACCATTCCT-3′; Runx1, 5′-AGCATGGTGGAGGTACTAGC-3′ and 5′-GGTCGTTGAATCTCGCTACC-3′; Runx3, 5′-ACCGCTTTGGAGACCTGCGCATG-3′ and 5′-CGCTGTAGGGGAAGGCGGCAGA-3′; BSP, 5′-AGGACTGCCGAAAGGAAGGT-3′ and 5′-ATGGAGACGGCGATAGTTCC-3′; and hypoxanthine-guanine phosphoribosyl transferase (HPRT), 5′-GCTGGTGAAAAGGACCTCT-3′ and 5′-CACAGGACTAGAACACCTGC-3′. Twenty cycles (for RANKL, OPG, Runx1, Runx3, and BSP) and 15 cycles (for HPRT) of amplification were carried out with a Gene Amp PCR system 2400 (30 s at 94°C, 30 s at 55–60°C, and 30 s at 72°C; Perkin Elmer). Amplified DNA was transferred to nylon membranes and hybridized with [32P]-labeled RANKL, OPG, Runx1, Runx3, BSP, and HPRT cDNA.

Cell culture

Chondrocytes were isolated from whole skeletons of Cbfa1−/− embryos at E18.5 as previously described(28) with minor modifications. In brief, minced cartilage tissues of Cbfa1−/− mice were treated with 0.125% trypsin and 0.1% EDTA in PBS for 1 h. After dissociation with collagenase solution (1.5 mg/ml) for 3.5 h, isolated chondrocytes were cultured in DMEM/Ham's F12 (1:1) hybrid medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS (Gibco BRL). Primary chondrocytes were plated at a density of 1 × 106 cells/dish in collagen-coated 60-mm plates (5 × 104 cells/cm2), and 2 days after seeding, cultures were stimulated for 5 days with each of the following factors: transforming growth factor (TGF) β1, insulin-like growth factor (IGF) 1, PDGF, interleukin (IL)-1β, IL-6, T3, and parathyroid hormone (PTH). Each culture medium was replaced every other day with fresh medium containing its particular factor. Cultures then were harvested for RNA preparation.

RESULTS

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

Lack of vascular invasion of cartilage in Cbfa1−/− mice

Because of the lack of vascular invasion of cartilage in Cbfa1−/− mice, VEGF expression was examined by immunohistochemical study (Fig. 1). In the femur of E18.5 wild-type mice, VEGF was expressed in prehypertrophic, hypertrophic, and terminal hypertrophic chondrocytes in the growth plates and in osteoblasts and endothelial cells in the bone marrow (Fig. 1A). In the femur of Cbfa1−/− mice, most of the cells were immature chondrocytes, which express type II collagen (Fig. 1B) but not type X collagen (Fig. 1C), and a small number of cells showed weak VEGF expression (Fig. 1D). However, in the tibia of Cbfa1−/− mice the diaphysis is composed of hypertrophic chondrocytes, which express type X collagen, and terminal hypertrophic chondrocytes, in which type X collagen expression is decreased (Fig. 1E). VEGF was expressed in prehypertrophic, hypertrophic, and terminal hypertrophic chondrocytes (Fig. 1F). The expression of VEGF in Cbfa1−/− mice also was confirmed by Northern blot analysis using RNA from distal limbs (data not shown). In addition, matrix in the diaphysis of Cbfa1−/− tibia was mineralized (Fig. 1E), indicating that chondrocytes in the diaphysis were fully mature.(11,14–16) These findings indicate that maturation into terminal hypertrophic chondrocytes and their VEGF expression are still insufficient for vascular invasion into cartilage.

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Figure FIG. 1. VEGF expression in Cbfa1−/− cartilage. Immunohistochemical study of (A) wild-type femur, (D) Cbfa1−/− femur, and (F) Cbfa1−/− tibia at E18.5 using anti-VEGF antibody and (G) E18.5 Cbfa1−/− tibia as a negative control without treatment with anti-VEGF antibody. In situ hybridization of E18.5 Cbfa1−/− femur using antisense probes of (B) type II collagen and (C) type X collagen and (E) E18.5 Cbfa1−/− tibia using an antisense probe of type X collagen. No signal was detected by in situ hybridization using sense probes of type II collagen and type X collagen (data not shown). Insets in panels A, B, and E show the skeleton of lower legs of (A) wild-type and (B and E) Cbfa1−/− mice stained with Alizarin red and Alcian blue, and the approximate part of each histological section is boxed. In Cbfa1−/− mice, (B and C) the entire femur is mainly composed of immature chondrocytes and (E) diaphysis of tibia is composed of hypertrophic chondrocytes and terminal hypertrophic chondrocytes with mineralization. (A) In femur of wild-type mice, VEGF is expressed in prehypertrophic, hypertrophic, and terminal hypertrophic chondrocytes. It is expressed also in osteoblasts and endothelial cells in bone marrow. (F) In Cbfa1−/− cartilage, VEGF is expressed mainly in prehypertrophic, hypertrophic, and terminal hypertrophic chondrocytes in tibia (bar = 100 μm).

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Chondrocyte maturation and vascular invasion of cartilage in spleen

To investigate the mechanisms of vascular invasion of cartilage, femurs from Cbfa1−/− mice, which completely lack osteoclasts,(11,16) were transplanted into spleens of normal mice. Before transplantation, most chondrocytes in Cbfa1−/− femurs were immature and expressed type II collagen but not type X collagen (Figs. 1B and 1C).(14,15) However, some chondrocytes in the diaphysis had matured into hypertrophic chondrocytes with mineralization by 1 week after transplantation (Figs. 2A, 2C, and 2E). In addition, vascular invasion appeared in the mineralized region, and TRAP+ cells were observed in the invaded region (Figs. 2E and 2G). Indeed, when we transplanted Cbfa1−/− tibias, in which the diaphyses were composed of hypertrophic chondrocytes (Fig. 1E), into spleens, vascular invasion occurred earlier than that in femurs (data not shown).

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Figure FIG. 2. Chondrocyte maturation and vascular invasion of Cbfa1−/− femurs transplanted into the spleen. Cbfa1−/− femurs at E18.5 were transplanted into spleens of normal mice and analyzed at (A, C, E, and G) 1 week and (B, D, F, H, I, and J) 2 weeks after transplantation. (A-D) H&E staining; (C and D) higher magnifications of boxed regions of panels A and B, respectively. (E and F) von Kossa staining; (G -I) TRAP staining; (I and J) higher magnification of (I) a TRAP+ cell and (J) immunohistochemical study using vacuolar H+-ATPase antibody. At 1 week after transplantation, (C) chondrocytes in the diaphysis are hypertrophic, (E) vascular invasion occurs in the mineralized region, and (G) TRAP+ cells are observed at the edge of the invaded region. At 2 weeks after transplantation, (B, D, and F) chondrocytes are hypertrophic and matrix is mineralized in most regions of the femur, (D and F) the diaphysis is occupied with blood vessels and hematopoietic cells, and (H) the surface of invaded cartilage is covered with TRAP+ cells, which are (I) multinucleated and (J) express vacuolar H+-ATPase. No signal was detected by immunohistochemistry in the absence of treatment with vacuolar H+-ATPase antibody (data not shown; panels A and B, bar = 1 mm; panels C-H, bar = 200 μm; panels I and J, bar = 10 μm).

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Two weeks after transplantation, most chondrocytes were hypertrophic and mineralization extended to the epiphysis (Figs. 2B, 2D, and 2F). In addition, most of the diaphysis was replaced by hematopoietic cells, and TRAP+ osteoclasts, which were multinucleated (Fig. 2I) and expressed vacuolar H+-ATPase (Fig. 2J), were observed on the surface of invaded cartilage (Fig. 2H). As controls, E14.5 wild-type tibias and femurs, which have a cartilaginous structure without vascular invasion, were transplanted into the spleen, because their developmental stage resembles stage E18.5 of Cbfa1−/− tibias (Figs. 1E and 4D). However, the entire process of chondrocyte maturation and vascular invasion in Cbfa1−/− transplants, including tibias and femurs, was severely retarded as compared with wild-type transplants (Figs. 2, 4D, and 4E). Therefore, these results indicate that the process of vascular invasion into Cbfa1−/− cartilage progressed but was still impaired in the spleen.

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Figure FIG. 4. Lack of osteoblast differentiation in Cbfa1−/− transplants. Osteoblast differentiation and bone formation in femurs were examined by in situ hybridization using antisense probes of (A and B) type I collagen and (F-H) osteocalcin, (C) a sense probe of type I collagen, and (D and E) by von Kossa staining. (A and C) E18.5 Cbfa1−/− femur at 1 week after transplantation. (B and H) E18.5 Cbfa1−/− femur at 2 weeks after transplantation. (D and F) E14.5 wild-type femur. (E and G) E14.5 wild-type femur at 1 week after transplantation. (A and B) At 1 week after transplantation of E18.5 Cbfa1−/− femur, type I collagen expression is observed in perichondrial cells and (B) at 2 weeks after transplantation, in an area invaded by blood vessels. (D) Bone formation is observed only in bone collar without vascular invasion of the femur in E14.5 wild-type mice, (E) but bone is generated in both trabecular and cortical regions at 1 week after transplantation of E14.5 wild-type femur. The sections were counterstained with nuclear fast red. (F) No osteocalcin expression is detected in E14.5 wild-type femur. (G) When E14.5 wild-type femur is transplanted into spleen, osteocalcin is highly expressed at 1 week after transplantation. (H) However, there is no osteocalcin expression in E18.5 Cbfa1−/− femur transplanted into the spleen for 2 weeks. No signal was detected in in situ hybridization using a sense probe of osteocalcin (data not shown; bars = 200 μm).

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Type X collagen and VEGF, which were expressed mainly in tibias (Figs. 1E and 1F) but not in femurs of Cbfa1−/− mice (Fig. 1C and 1D),(14) were detected in hypertrophic chondrocytes of femurs 1 week after transplantation (Fig. 3A). Although osteopontin, BSP, and MMP-13 were barely detectable in the terminal hypertrophic chondrocytes of Cbfa1−/− mice,(14) they were strongly expressed in the terminal hypertrophic chondrocytes after transplantation (Fig. 3A). Their expression was confirmed also by Northern blot analysis (Fig. 3B). Expression of RANKL was much less in E18.5 Cbfa1−/− skeleton than in E14.5 wild-type skeleton, but it was strongly induced after transplantation in Cbfa1−/− transplants (Fig. 3C). In contrast, the expression of OPG in E18.5 Cbfa1−/− skeleton was much greater than that in E14.5 wild-type skeleton, and it was suppressed after transplantation in Cbfa1−/− transplants. Further, the other runt-domain transcription factors Runx1 and Runx3 showed slight and strong induction, respectively, in Cbfa1−/− transplants (Fig. 3C).

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Figure FIG. 3. Induction of type X collagen, VEGF, osteopontin, BSP, MMP13, RANKL, Runx1, and Runx3 in Cbfa1−/− transplants. (A) In situ hybridization and immunohistochemistry. At 1 week after transplantation of E18.5 Cbfa1−/− femur, chondrocyte maturation was examined by in situ hybridization using antisense (left side) and sense (right side) probes of type II collagen [α1(II)], type X collagen [α1(X)], osteopontin (OP), BSP, and MMP-13, and by immunohistochemical study using anti-VEGF antibody. In the diaphysis of the femur, type II collagen expression is suppressed and type X collagen expression is up-regulated. VEGF is expressed in prehypertrophic, hypertrophic, and terminal hypertrophic chondrocytes and endothelial cells. Expression of OP, BSP, and MMP-13 is limited in the terminal hypertrophic chondrocytes surrounding vascular invasion (bar = 200 μm). (B) Induction of OP, BSP, and MMP-13 expression in Cbfa1−/− transplants. The expression of OP, BSP, and MMP-13 was examined by Northern blot analysis using RNA from Cbfa1−/− and wild-type skeletons, transplants, and wild-type spleen. (C) Induction of RANKL, Runx1, and Runx3 in Cbfa1−/− transplants. The expression of RANKL, OPG, Runx1, and Runx3 was examined by RT-PCR. (B and C) 1, skeleton of Cbfa1−/− embryo at E18.5; 2, grafted E18.5 Cbfa1−/− femurs at 1 week after transplantation; 3, skeleton of wild-type embryo at E14.5; 4, grafted E14.5 wild-type femurs at 1 week after transplantation; 5, wild-type spleen. Hybridization with the GAPDH probe and RT-PCR of HPRT were used, respectively, as internal controls for Northern blot analysis and RT-PCR analysis.

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Lack of osteoblast differentiation and bone formation in Cbfa1−/− cartilage in spleen

The matrix around hypertrophic chondrocytes in Cbfa1−/− femurs was severely mineralized after transplantation (Figs. 2E and 2F). However, neither bone collar nor trabeculae were formed, and no mature osteoblasts were observed morphologically for 3 weeks after transplantation (Fig. 2 and data not shown). To investigate the possibility that some of the calcified matrix seen in femurs after transplantation was generated by osteoblasts, the expression of osteoblastic markers type I collagen and osteocalcin was examined. Mesenchymal cells surrounding the cartilage of Cbfa1−/− limb skeletons express type I collagen.(14) They also were present in the perichondrial region of Cbfa1−/− femurs at 1 week after transplantation, and some of them were observed in diaphysis invaded by blood vessels at 2 weeks after transplantation (Figs. 4A and 4B). When we transplanted E14.5 wild-type femurs into spleens, blood vessels invaded the femurs and trabeculae were formed at 1 week after transplantation (Figs. 4D and 4E). Although osteocalcin-positive cells were absent in E14.5 wild-type femurs, many osteoblastic cells in trabecular and cortical regions of the femurs expressed osteocalcin 1 week after transplantation (Figs. 4F and 4G). However, no osteocalcin-positive cells were present in Cbfa1−/− femurs for 3 weeks after transplantation (Fig. 4H and data not shown). These results indicate that type I collagen-positive perichondrial mesenchymal cells in Cbfa1−/− mice failed to differentiate into mature osteoblasts in spleen.

Induction of osteopontin and MMP-13 expression in Cbfa1−/− chondrocytes in vitro

To investigate factors in the spleen that stimulate the expression of osteopontin, BSP, and MMP-13, chondrocytes from Cbfa1−/− skeleton were cultured in vitro in the presence of each of the various local factors TGF-β1, IGF-I, PDGF, IL-1β, and IL-6 and hormones (T3 and PTH) that are considered to be present in the spleen. The expression of osteopontin and MMP-13 was examined by Northern blot analysis (Fig. 5A). TGF-β1, PDGF, IL-1β, and T3 induced both osteopontin and MMP-13 expression, whereas IL-6 induced only weak osteopontin expression. BSP expression was examined by RT-PCR analysis because of its weak expression (Fig. 5B). All of the factors and hormones examined failed to induce BSP, and PDGF, IL-1β, and PTH suppressed BSP expression in a dose-dependent manner. These findings indicate that the factors present in the spleen are able to induce at least osteopontin and MMP-13 expression independent of Cbfa1.

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Figure FIG. 5. Induction of osteopontin, MMP-13, and BSP expression in Cbfa1−/− chondrocytes in vitro. (A) Induction of osteopontin and MMP-13 expression was examined by Northern blot analysis, and (B) induction of BSP expression was analyzed by RT-PCR. Cbfa1−/− chondrocytes were cultured for 5 days in the presence or absence of each of the following factors: TGF-β1, IGF-1, PDGF, IL-1β, IL-6, T3, and PTH. Concentrations of factors are indicated. Hybridization with the GAPDH probe and RT-PCR of HPRT were used, respectively, as internal controls for Northern blot analysis and RT-PCR analysis. Similar results were obtained in two or three independent experiments.

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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

To investigate the mechanisms of vascular invasion of cartilage and the role of the hematopoietic organ in endochondral ossification, Cbfa1−/− femurs were transplanted into the spleen. After transplantation, immature chondrocytes in the Cbfa1−/− femurs matured into terminal hypertrophic chondrocytes; the expression of osteopontin, BSP, and MMP-13 was up-regulated in the terminal hypertrophic chondrocytes; and blood vessels invaded the cartilage with osteoclasts. Further, TGF-β1, PDGF, IL-1β, and T3 induced the expression of osteopontin and MMP-13 in Cbfa1−/− chondrocytes in vitro. However, the entire process of chondrocyte maturation and vascular invasion in Cbfa1−/− transplants was severely retarded. These findings show that the hematopoietic organ is able to support vascular invasion of cartilage in a Cbfa1-independent manner but does so less effectively without Cbfa1, indicating that Cbfa1 and factors in the hematopoietic system, including growth factors, cytokines, hormones, and proteinases, coordinate in regulating the process of vascular invasion.

Cbfa1−/− mice showed disturbed chondrocyte maturation, and most of the chondrocytes were resting or proliferating.(14,15) Cbfa1 induced chondrocyte maturation in vitro,(17) and Cbfa1 and dominant negative Cbfa1 transgenic mice controlled by type II collagen promoter and enhancer showed, respectively, accelerated and decelerated chondrocyte maturation.(18) Therefore, Cbfa1 is required for chondrocyte maturation. As indicated by these studies, chondrocyte maturation was severely retarded after transplantation into the spleen. However, our results also indicated that factors from the hematopoietic organ weakly supported Cbfa1-independent chondrocyte maturation. As chondrocytes matured in restricted parts of the skeletons of Cbfa1−/− mice, including tibia, fibula, radius, and ulna, factors from the perichondrial region appeared to have limited ability to support chondrocyte maturation in a Cbfa1-independent manner. However, chondrocyte maturation was not observed in whole skeleton, including tibia, fibula, radius, and ulna, in dominant negative Cbfa1 transgenic mice, in which all runt-domain factors had been suppressed in chondrocytes.(18) Therefore, it is possible that other runt-domain factors also have an ability to induce chondrocyte maturation. The other runt-domain genes Runx1 and Runx3 are expressed also in chondrocytes,(29) and Runx1 and Runx3 showed slight and strong induction, respectively, in Cbfa1−/− transplants (Fig. 3C). Thus, they may be involved in chondrocyte maturation, induction of osteopontin, BSP, and MMP-13, and vascular invasion in Cbfa1−/− transplants. Because Cbfa1 is highly expressed in terminal hypertrophic chondrocytes,(14,15) which are in close contact with bone marrow, the induction of runt-domain factors in the terminal hypertrophic chondrocytes by the hematopoietic system seems to be important for the process of growth plate vascularization.

Cbfa1−/− mice completely lacked vascular invasion of calcified cartilage, irrespective of the expression of VEGF and down-regulation of chondromodulin I (Fig. 1F).(14) However, transplantation of Cbfa1−/− cartilage into spleen resulted in the expression of osteopontin, BSP, and MMP-13, the appearance of osteoclasts, and vascular invasion of cartilage. The expression of osteopontin and BSP will work positively in the vascular invasion of cartilage, because osteopontin and BSP, which share the same small cell attachment motif (Arg-Gly-Asp [RGD]) recognized by integrins, are considered to promote the attachment of osteoclasts to the extracellular matrix.(30) The expression of MMP-13, which preferentially cleaves type II collagen over type I and III,(31) would have supported the vascular invasion of Cbfa1−/− cartilage in the spleen. Indeed, the appearance of osteoclasts, which express MMP-9, would have accelerated the vascular invasion because MMP-9−/− mice showed a delay in growth plate vascularization.(8) The appearance of osteoclasts also indicates that monocytic hematopoietic cells are able to differentiate into osteoclasts in the absence of osteoblasts, probably because of the induction of RANKL, osteopontin, and BSP and the suppression of OPG in Cbfa1−/− transplants. Therefore, our findings show that the induction of MMP-13, the appearance of osteoclasts, and the promoted attachment of osteoclasts to the mineralized cartilage matrix supported the vascular invasion of Cbfa1−/− cartilage but are still not sufficient for efficient vascular invasion.

TGF-β1, PDGF, IL-1β, and T3 induced both osteopontin and MMP-13, and IL-6 weakly induced osteopontin in Cbfa1−/− chondrocytes (Fig. 5A). We reported that Cbfa1 and Ets1 cooperatively regulate osteopontin expression,(32) and osteopontin expression in hypertrophic chondrocytes in Cbfa1−/− mice virtually was absent.(11,14) Therefore, our data indicate that osteopontin expression is regulated by two pathways, one that is Cbfa1-dependent and one that is Cbfa1-independent. In addition, we and others have shown that Cbfa1 is involved in the transcriptional activation of MMP-13(25,33) and the PTH-mediated transcriptional activation of MMP-13.(34,35) In accordance with these reports, PTH failed to induce MMP-13 in the absence of Cbfa1 (Fig. 5A). Although both Cbfa1 and AP-1 have been shown to be required for the transcriptional activation of MMP-13,(25,33,34) our findings show that the induction of MMP-13 expression by TGF-β1, PDGF, IL-1β, and T3 is regulated, at least in part, through a Cbfa1-independent pathway. However, the synergistic effects of these factors with Cbfa1 on osteopontin and MMP-13 expression need to be investigated further. We found that PDGF, IL-1β, and PTH suppressed BSP expression in the absence of Cbfa1, but we failed to find factors that induce BSP expression independent of Cbfa1, although TGF-β1 and PTH simulated BSP expression in chicken primary osteoblasts and an osteosarcoma cell line (ROS 17/2.8), respectively.(36,37)

Our findings indicate that the hematopoietic system is able to induce vascular invasion independent of Cbfa1. However, the retarded vascular invasion in Cbfa1−/− transplants indicates that Cbfa1 and factors in the hematopoietic system, including growth factors, cytokines, hormones, and proteinases, coordinate in regulating the process of vascular invasion of cartilage, suggesting an important cooperation between Cbfa1 and factors from bone marrow in the process of growth plate vascularization. The results of our study also indicate that factors from the hematopoietic organ are not sufficient for vascular invasion and that gene regulation by Cbfa1 in terminal hypertrophic chondrocytes is required for efficient vascular invasion. Thus, the genes regulated by Cbfa1 in terminal hypertrophic chondrocytes need to be investigated further.

Acknowledgements

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

We thank M. Iwamoto and M. Enomoto-Iwamoto for critically reading this article, Y. Moriyama for vacuolar H+-ATPase antibody, N. Udagawa for technical advice, M. Inada for technical assistance, R. Hiraiwa for maintaining mouse colonies, and M. Yanagita for secretarial assistance. This work was supported by grants from the Ministry of Education, Science and Culture, Japan, and the Tokyo Biochemical Research Foundation.

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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