Progressive increase in bone mass and development of odontomas in aging osteopetrotic c-src-deficient mice

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Progressive Increase in Bone Mass and Development of Odontomas in Aging Osteopetrotic c-src-deficient Mice M. AMLING,1,2 L. NEFF,2 M. PRIEMEL,1 A. F. SCHILLING,1 J. M. RUEGER,1 and R. BARON2,3 Department of Trauma and Reconstructive Surgery, Hamburg University School of Medicine, Hamburg, Germany 2 Departments of Cell Biology and Orthopedics,3 Yale University School of Medicine, New Haven, CT, USA

it has been shown that the defect responsible for the osteopetrotic phenotype of the src⫺/⫺ mouse is cell autonomous and occurs in mature OCs.5,24 The specific signaling pathways that require c-src expression for normal osteoclast activity have only partially been elucidated. We have previously reported that the protooncoprotein c-Cbl4,40 is one of the downstream signaling elements that require c-src for tyrosine phosphorylation and is in turn also necessary for osteoclast function.39 However, the longterm effects of the lack of c-src and impaired osteoclast function on the skeleton as well as the consequences of an arrest of bone resorption, particularly on the regulation of bone formation and remodeling, are not known. This question is of particular importance because evolution of the osteopetrotic phenotypes varies among the various known natural or transgenic mutants. Both impaired osteoclast differentiation and impaired osteoclast function result in osteopetrosis.3,10,30,32,36 Much of our current understanding of the mechanisms of bone resorption stems from studies analyzing patients and animals with osteopetrosis.10,30,32,36 At present, there are eight described mutations that result in an osteopetrotic phenotype in the mouse, seven of which are in identified genes, whereas the gene mutated in the grey lethal (gl/gl)14 mouse model remains unknown. The first described osteopetrotic model is caused by a point mutation in the CSF-1 gene in the op/op mouse.9,22,47,48 In contrast to src⫺/⫺ mice, in which osteoclast function rather than osteoclast differentiation is impaired, the lack of functional, active CSF-1 in op/op mice results in a block of osteoclast differentiation, as do disruptions of fos,15,16,45 PU1,44 and NFKB.6,11 In contrast, mutations of a basic leucine zipper/helixloop-helix transcription factor in the micropthalmia locus17 and the 116 kDa subunit of the vacuolar proton pump in the oc/oc mouse34,35,48 also lead, like deletion of src, to osteopetrosis by altering osteoclast function but not their differentiation, as does the mutation in the currently unidentified gene in the grey lethal mouse.14 Whereas op/op osteopetrosis reverses with age,9,22 other mutants, such as the fos knockout, continue to worsen with time,16 while nothing is yet known about others. Early morphological observations of bones of src⫺/⫺ mice5,24 have shown that osteoclasts differentiate properly in the absence of c-src, but that c-src is required for mature osteoclasts to form ruffled borders and resorb bone, and that this functional defect is cell autonomous. However, studies on the long-term physiological consequence of c-src deletion were hampered by the fact that src⫺/⫺ mice die prematurely, due to starvation in the absence of erupted teeth. In this study we report that aging src⫺/⫺ mice maintained on

The critical role of c-src in osteoclast-mediated bone resorption has been emphasized by gene deletion experiments in mice. However, the long-term effects of the lack of c-src and impaired osteoclast function on the skeleton remain unknown. To further study the physiological role of c-src and to circumvent the early death of srcⴚ/ⴚ mice, due to starvation in the absence of erupted teeth, we maintained mice on a liquid diet. At the age of 2 months the srcⴚ/ⴚ mice presented signs of airway obstruction and all mice died progressively between 2.5 and 6 months of age. Radiography demonstrated severe osteopetrosis of the whole skeleton. Histomorphometrical analysis of the srcⴚ/ⴚ mice confirmed a significant increase in bone mass with age, resulting in complete loss of bone marrow spaces in some bones and explaining the consistent hepatosplenomegaly, due to extraskeletal hematopoesis. Histopathological examination of the skull revealed the presence of odontomas in the region of the unerupted incisors, with a penetrance of 100% in the aging srcⴚ/ⴚ mice. Although odontomas are benign lesions, their progressive growth leads to the obliteration of the nasal airways, progressive suffocation, and death in srcⴚ/ⴚ mice. These results suggest that: (i) in the absence of bone resorption, bone formation continues and leads to progressive accentuation of the osteopetrotic phenotype in srcⴚ/ⴚ mice; (ii) osteoclastic function is required for regular eruption of the incisors and deficient bone resorption is associated with the development of odontomas; and (iii) srcⴚ/ⴚ mice die by suffocation due to airway obliteration as a result of progressive odontoma growth. (Bone 27:603– 610; 2000) © 2000 by Elsevier Science Inc. All rights reserved. Key Words: c-src; Osteoclast; Bone resorption; Osteopetrosis; Odontoma. Introduction c-src was first identified as the cellular counterpart of the transforming protein of Rous sarcoma retrovirus, v-src.13,21 The primary defect in mice lacking the c-src gene is osteopetrosis, resulting from a deficiency in bone resorption by osteoclasts (OCs).25,38 OCs express high levels of the c-src protein,19,41 and Address for correspondence and reprints: Dr. Roland Baron, Departments of Cell Biology and Orthopedics, Yale University School of Medicine, 333 Cedar Street, SHM IE-55, New Haven, CT 06510. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.

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a liquid diet to ensure survival have, unlike op/op mice, no spontaneous tendency to correct their skeletal defect. Instead, these mice show a progressive accentuation of the osteopetrotic phenotype, thereby demonstrating a continued imbalance between bone resorption and bone formation, leading to a protracted increase in bone mass. This further establishes that there is no obligatory cross control between bone formation and bone resorption, and that bone formation is not tightly controlled by bone resorption and/or bone mass. In addition, we observed that continued impaired osteoclastic bone resorption is associated with failure of incisor tooth eruption and the development of odontomas in all src⫺/⫺ mice. This ultimately causes their death by suffocation due to airway obliteration as a result of progressive odontoma growth. Materials and Methods Animals The src⫺/⫺ mice were obtained from Dr. P. Soriano38 and maintained on a liquid diet (LD82, Bio-Serv, Holton Industries Co., Frenchtown, NJ) and crushed food after weaning (day 14). Wild-type littermates maintained on the same type of diet were used as controls. All mice received tap water ad libitum and were kept at 75°F. A total of 30 src⫺/⫺ mice were maintained until they died between 2.5 and 6 months of age. Sample Preparation After whole-animal contact radiography (Faxitron X-ray Cabinet, Faxitron Co., Wheeling, IL) and autopsy, bone samples (skull, spine, tibiae, femurs, humeri) were dissected out and fixed in 3.7% buffered formalin for 18 h at 4°C. After dehydration the undecalcified bones were embedded in methylmethacrylate and 5-␮m-thick sections were prepared on a rotation microtome (Cut 4060E, MicroTech, Munich, Germany) as previously described.1,2 Sections were stained with toluidine blue, von Kossa, or Goldner trichrome and evaluated on a Zeiss Axioskop microscope (Carl Zeiss, Jena, Germany). Histomorphometry

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spongiosa of the metaphysis. The slices were fixed in PLP for an additional 4 h at 4°C and then washed in phosphate-buffered saline (PBS) containing 10% dimethylsulfoxide (DMSO) as a cryoprotectant. The tissue was subsequently quick frozen and 40 ␮m sections were prepared on a cryostat (Bright Instruments Co., Huntingdon, UK) using tungsten carbide knives. Sections were incubated overnight at 4°C in MAb p31, a monoclonal antibody generated in our laboratory against the 31 kDa subunit of the vacuolar proton pump diluted in PBS and 0.1% bovine serum albumin (BSA). After washing (2 h) in PBS ⫹ 0.1% BSA, the sections were incubated with Fab fragments of perioxidatelabeled goat anti-mouse immunoglobulin G (IgG; Biosys, France). Sections were incubated with the secondary antibody at a dilution of 1:100 in PBS ⫹ 0.1% BSA for 2 h at 20°C. After washing, the sections were reacted with DAB (1 mg/mL in 0.05 Tris-buffer [pH 7.4]; Polysciences, Inc., Warrington, PA) in the presence of 0.1% H2O2, and postfixed in ferrocyanide-reduced OsO4. After embedding in Epon (Polybed 812, Polysciences, Inc.), 1 ␮m sections were cut with a diamond knife and stained with lead citrate. Grids were viewed on a Jeol-CX 100 electron microscope (Jeol, Inc., Peabody, MA). Results Extended Survival of src⫺/⫺ Mice Maintained on Liquid Diet The src⫺/⫺ mice initially survived without gross abnormalities, aside from the previously described changes. However, starting at the age of 2 months, the animals showed signs of airway obstruction and all died progressively between 2.5 and 6 months of age. At 3 weeks of age, the src⫺/⫺ mice had the same body weight, skeletal size, skull size as the intact controls. At 4 and 6 months, the src⫺/⫺ mice had a significantly decreased body weight as compared with controls, whereas the overall bone length of the femur and lumbar spine and size of skull were not altered significantly as compared with the respective skeletal sites of the control littermates (Figure 1). A distended gastrointestinal tract (GI), hepatosplenomegaly, and deformations of the snout were consistent findings at autopsy, the latter being associated with radiodense tumoral masses of the jaws (see later).

Quantitative histomorphometry was performed on toluidine blue-stained, undecalcified proximal tibia sections from 3-, 16-, and 24-week-old mice, respectively. For comparative histomorphometry six control and six src⫺/⫺ animals were used at the ages of 3 and 16 weeks, respectively, whereas each three animals were used at the age of 24 weeks for each group. Analyses of bone volume (BV/TV, %), trabecular thickness (Tb.Th, ␮m), trabecular number (Tb.N, /mm), trabecular separation (Tb.Sp, ␮m), osteoblast surface per bone surface (Ob/BS, %), osteoblast number per bone perimeter (N.Ob/ B.Pm, /mm), osteoclast surface per bone surface (Oc/BS, %), and osteoclast number per bone perimeter (N.Oc/B.Pm, /mm), were carried out according to ASBMR standards31 using Osteomeasure histomorphometry system (Osteometrix, Atlanta, GA) as previously described.2 Statistical analysis was performed using the unpaired Student’s t-test. p ⬍ 0.05 was considered statistically significant.

Progressive Osteopetrosis in src⫺/⫺ Mice

Ultrastructural Analysis

Histomorphometry in src⫺/⫺ Mice Shows a Progressive Increase in Bone Mass

For ultrastructural examination of osteoclasts animals were perfused with 2% paraformaldehyde, lysine (0.75 mol/L), and sodium periodate (0.01 mol/L) (PLP) for 5 min. The distal femurs were dissected out and slices were cut out of the primary

Radiography and histomorphometry demonstrated severe osteopetrosis of the whole skeleton. Although the severity of osteopetrosis varied within the same animal, with a marrow cavity being present in some bones, diaphyseal marrow space was almost completely filled with bone in the majority of bones in all src⫺/⫺ animals (Figures 2 and 3). Interestingly, while trabecular bone volume declined slightly from week 3 to week 16 and then remained relatively constant in control mice, it increased progressively in the absence of c-src. Gender-specific differences were not observed. The progressive increase in bone volume in src⫺/⫺ animals was due mainly to a significant increase in the number of trabecular elements (Tb.N) and, consequently, a strikingly reduced trabecular separation (Tb.Sp), whereas the thickness of trabeculae (Tb.Th) did not differ significantly between src⫺/⫺ animals and normal controls (Table 1).

Osteoclast number was significantly higher (threefold) in src⫺/⫺ mice as compared with controls (Table 1 and Figure 4). The bone surface covered by osteoclasts was even more markedly increased

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Figure 1. Whole-skeleton contact X-ray of a 6-month-old wild-type mouse on the left and a 6-month-old src⫺/⫺ mouse on the right (a). The overall increase in mineral density of the whole skeleton in src⫺/⫺ mice as well as the radiodense masses at the snout, within the vertebral bodies throughout the spine, and the metaphyses of long bones (as indicated by arrows) can be seen. Although src⫺/⫺ mice are grossly similar to control littermates at 3 weeks, their body weight (b) is decreased at 4 and 6 months, whereas their overall bone length [(c) skull, (d) femur, (e) lumbar spine] is not altered significantly. Error bars indicate standard deviation. *Significant at p ⬍ 0.05.

due to the extended shape of src⫺/⫺ osteoclasts. Although src⫺/⫺ mice osteoclasts were in tight contact with the bone surface, they did not form a ruffled border (Figure 5). Interestingly, the fact that bone mass continued to increase demonstrates that, although the relative bone surface covered by osteoblasts and the total number of osteoblasts per unit bone surface were significantly reduced in src⫺/⫺ animals, bone formation was continuously exceeding bone resorption in the absence of c-src.

examination of the skull revealed the presence of odontomas in the region of the unerupted incisors, with a penetrance of 100% in aging src⫺/⫺ mice (Figure 6). They presented as early tooth anlagen, progressively increasing in number with expansive growth within the upper and lower jaw, widening and deforming the jaws, and finally leading to occlusion of the nasal airways. The odontomas were restricted to the incisors, whereas the molar teeth, the growth of which is limited over time, were unaffected.

Odontoma Development in Aging src⫺/⫺ Mice

Discussion

Radiographically, the lesions of the jaws were detectable in src⫺/⫺ mice as early as 3 weeks of age. Histopathological

This study shows that, despite the arrest of bone resorption and a dramatic increase in bone mass, bone formation continued to

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Figure 2. Contact X-ray of the lumbar spine in wild-type and src⫺/⫺ mice. Note the increased bone density in 16- and 24-week-old src⫺/⫺ animals as compared with the spines of the 16-week-old wild-type controls on the right.

exceed bone resorption in src⫺/⫺ mice. In the absence of c-src osteoclasts were functionally impaired and did not form a ruffled border.5,24 The src-deficient osteoclast attached tightly to the bone surface, as demonstrated by electron microscopy, and

showed a reorganization of their attachment apparatus,27,28 from a motile toward a more stably attached phenotype.29 Interestingly, the number of osteoclasts was significantly increased in src⫺/⫺ mice. Whether the latter observation represents a direct

Figure 3. Histology of the tibia from 3- and 16-week-old animals demonstrating progressive osteopetrosis in src⫺/⫺ animals. (undecalcified preparation, 5 ␮m, toluidine blue staining).

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Table 1. Histomorphometrical analysis of tibiae from src⫺/⫺ and wild-type mice during aging (mean ⫾ SD) BV/TV (%)

Tb.Th (␮m)

Tb.N (/mn)

Tb.Sp (␮m)

Ob.S/BS (%)

N.Ob/B.Pm (/mm, mm2)

Oc.S/BS (%)

N.Oc/B.Pm (/mm, mm2)

src⫺/⫺ 3 weeks (n ⫽ 6) 33.61a ⫾ 3.28 12.73 ⫾ 2.03 26.80a ⫾ 3.31 25.01a ⫾ 2.60 31.60a ⫾ 11.61 20.29a ⫾ 4.41 10.49a ⫾ 2.52 3.99a ⫾ 0.85 16 weeks (n ⫽ 6) 48.39a ⫾ 8.80 45.51 ⫾ 14.41 11.28a ⫾ 3.28 48.06 ⫾ 12.38 8.83a ⫾ 1.09 4.50a ⫾ 0.71 6.70a ⫾ 5.77 3.56a ⫾ 1.31 24 weeks (n ⫽ 3) 57.45a ⫾ 5.68 39.25 ⫾ 6.62 14.74a ⫾ 0.93 28.77a ⫾ 2.27 7.45a ⫾ 0.97 3.99a ⫾ 0.40 7.74a ⫾ 4.13 3.80a ⫾ 1.09 src⫹/⫹ 3 weeks (n ⫽ 6) 16.34 ⫾ 3.52 13.49 ⫾ 1.19 12.11 ⫾ 2.41 71.68 ⫾ 16.78 46.09 ⫾ 8.55 37.23 ⫾ 5.67 2.35 ⫾ 1.13 1.16 ⫾ 0.51 16 weeks (n ⫽ 6) 12.63 ⫾ 0.98 31.26 ⫾ 8.32 4.23 ⫾ 0.92 216.17 ⫾ 54.55 29.36 ⫾ 9.21 23.48 ⫾ 7.31 2.02 ⫾ 1.94 0.96 ⫾ 0.90 24 weeks (n ⫽ 3) 9.98 ⫾ 1.01 33.52 ⫾ 11.47 3.14 ⫾ 0.75 297.76 ⫾ 67.97 33.28 ⫾ 5.85 26.86 ⫾ 3.96 2.96 ⫾ 1.07 1.43 ⫾ 0.66 See Materials and Methods for abbreviations of bone parameters. n is number of animals in each group. p ⫽ 0.05.

a

Figure 4. Progressive osteopetrosis and increased osteoclast number in src⫺/⫺ mice. Error bars indicate standard deviation. Number (n) of animals in each group: n ⫽ 6 animals (3 weeks), n ⫽ 6 animals (16 weeks), n ⫽ 3 animals (24 weeks).

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Figure 5. Electron-microscopic images of osteoclasts from wild-type and src⫺/⫺ animals after immunolabeling for the p31 subunit of the vacuolar proton pump. (a) The wild-type osteoclast seals a resorbing compartment and forms an extensive ruffled border, which is strongly labeled by p31 due to an accumulation of the vacuolar proton pump. (b) In contrast, in the absence of c-src, osteoclasts attach tightly to the bone surface and the cell does not form a ruffled border. Also, there is no proton-pump accumulation.

effect of c-src on osteoclast differentiation, or is an indirect result of decreased bone resorption in the absence of c-src remains to be determined. Histomorphometrical analysis of src⫺/⫺ mice confirmed a significant increase in bone mass with age, resulting in complete loss of bone marrow spaces in some bones and explaining the consistent hepatosplenomegaly, probably due to extraskeletal hematopoesis. Progressive osteopetrosis implies continuing bone formation in the presence of osteoclasts, which were functionally impaired. This positive balance between bone formation and bone resorption toward bone formation is even more striking in view of the fact that osteoblast numbers per unit bone surface were lower in src⫺/⫺ mice as compared with control littermates. However, when taking into account the markedly increased bone density, the absolute number of osteoblasts per bone tissue area (N.Ob/T.Ar) was significantly increased in src⫺/⫺ mice (N.Ob/ T.Ar: controls wt 712.2 ⫾ 46 vs. src⫺/⫺ 834.6 ⫾ 74, at 3 weeks). Whether this observation can be attributed to a direct effect of c-src on osteoblast function remains to be determined.42 Although decreased resorption in the growth area could play a role in the increased bone mass of src⫺/⫺ mice, the fact that bone mass continued to increase while bone growth decreased in older animals clearly demonstrates that remodeling was affected. This was further confirmed by the results of in vitro studies, which

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demonstrated clearly a cell-autonomous increase in osteoblast differentiation in the absence of src.42 The maintenance of a nearly constant bone mass throughout bone remodeling and the requirement for an osteoclast differentiation factor synthesized by osteoblast progenitors23,37,49 have led to the hypothesis that bone resorption and bone formation are functionally controlled by one another.33 However, several recent in vivo observations have challenged this hypothesis. First, we have previously generated an inducible transgenic osteoblast ablation model and showed that, despite a complete arrest of bone formation and a dramatic decrease in bone mass, bone resorption remains unaffected in these mice.7 Second, in c-fosdeficient mice that have no osteoclasts, osteopetrosis suggests that bone formation is not arrested, even though there is no bone resorption.45 Third, several human skeletal disorders summarized as osteosclerosis have been marked by an increase in bone formation, without evidence of a parallel increase in bone resorption.46 Taken together, these observations and results from the present study indicate that bone formation continues in the absence of functionally active osteoclasts and, despite a continued increase in bone mass in src-deleted mice, show that there is no obligatory tight functional cross control between resorption and formation of bone to regulate bone mass. In their original study Soriano and colleagues noted changes in the jaws of src⫺/⫺ mice and described the formation of pseudo-odontomas in the proximity of some molar teeth.38 However, the nature of these lesions and their significance during the course of skeletal growth and aging remained unknown. Recently, Tiffee et al.43 described dental abnormalities in 4-weekold src⫺/⫺ and op/op mice. They examined the morphology of the jaws in great detail and found that the roots of the incisors were distorted by foci of haphazard proliferation of odontogenic epithelium associated with primitive tooth structures resembling the early stages of odontomas. Although odontomas are benign lesions,8,12,18,26 their progressive growth leads to the obliteration of the nasal airways, progressive suffocation, and death in src⫺/⫺ mice, explaining the distended gastrointestinal tract. The mechanisms underlying the development of odontomas are still unclear. However, because odontomas have also been reported in the toothless rat20 and the op/op mouse,43 our finding strongly suggests that impaired osteoclastic bone resorption may be responsible for the development of these tumors—at least in rodents, where the incisors continue to erupt at very high rate through life. One can speculate that it is the imbalance between continued growth of the incisor’s apex and the impaired resorption that leads to changes in tooth anlagen proliferation and growth, leading to development of odontomas. Studies of osteoclast function in patients with odontomas could therefore be of special interest for testing this hypothesis. In conclusion, maintenance of src⫺/⫺ mice on a liquid diet allows for further study of the physiological role of c-src in skeletal physiology. This study has demonstrated that an imbalance between osteoclastic function and the tooth eruption process is associated with the development of odontomas, which ultimately result in death in src⫺/⫺ mice by suffocation due to airway obliteration. Furthermore, the lack of c-src is not compensated for during aging and src⫺/⫺ mice have no spontaneous tendency to recover, unlike op/op mice, but instead show a progressive accentuation of the osteopetrotic phenotype. The increasing severity of osteopetrosis indicates that, in src⫺/⫺ mice, bone formation continues despite only slight bone resorption. This further establishes that bone resorption and/or bone mass do not closely control the extent of bone formation in vivo in mice.

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Figure 6. Odontomas in src⫺/⫺ mice. Contact X-rays of the skull from src⫺/⫺ mice showing radiodense tumors of the jaws as early as 3 weeks after birth (arrow). These tumors, which started from the region of the unerupted incisors, increased progressively with age (16 and 24 weeks), leading to occlusion of the nasal airways, and were histologically identified as odontomas. There is a histological appearance of masses of small misshapen teeth and a relatively high degree of differentiation, which is typical for compound odontoma-like lesions (undecalcified preparation, 5 ␮m, toluidine blue staining).

Acknowledgments: The authors thank Karen Ford for help in maintaining the src⫺/⫺ colony. M. A. thanks G. Delling for his early support and S. Tanaka for his generosity and useful advice. This work was supported by NIH Grant # AR42927 to R. B. and by DFG Grant Am 103/2-1 to M. A. A. F. S. is a fellow of the German Research Community DFG Graduiertenkolleg 476.

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Date Received: March 27, 2000 Date Revised: July 17, 2000 Date Accepted: July 17, 2000