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Trabecular bone mass and bone formation are preserved after limb immobilisation in p53 null mice
  1. R Okazaki1,
  2. A Sakai2,
  3. A Ootsuyama1,
  4. T Sakata2,
  5. T Nakamura2,
  6. T Norimura1
  1. 1Department of Radiation Biology and Health, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
  2. 2Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
  1. Correspondence to:
    Dr A Sakai
    Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan; a-sakaimed.uoeh-u.ac.jp

Abstract

Objectives: To determine whether disruption of the p53 gene leads to preservation of trabecular bone volume (BV) after limb immobilisation.

Materials and methods: Tibias of immobilised hind limbs of p53 gene knockout (p53−/−) and wild-type (p53+/+) mice were compared. Right knee joints of 8 week old mice were immobilised in full extension for 7 days. Trabecular structure and bone formation were similar in the p53−/− and p53+/+ control groups.

Results: Immobilisation significantly reduced BV to 77% of the control in p53+/+ mice, but no change was noted in p53−/− mice. After immobilisation, bone formation rate was significantly reduced in p53+/+ but not in p53−/− mice. In bone marrow cell cultures the numbers of alkaline phosphatase positive colony forming units-fibroblastic and mineralised nodules were significantly reduced by immobilisation in p53+/+ but not in p53−/− mice. Immobilisation enhanced p53 mRNA expression in marrow cells of p53+/+ mice and increased terminal dUTP nick end labelling positive osteocytes and marrow cells in p53+/+ but not in p53−/− mice. Lack of p53 in immobilised mice was associated with preservation of trabecular bone mass and bone formation and suppression of significant apoptosis of marrow cells.

Conclusion: Disruption of the p53 gene preserves trabecular bone mass and leads to bone formation after limb immobilisation.

  • apoptosis
  • p53
  • bone marrow
  • colony forming units
  • immobilisation
  • AP, alkaline phosphatase
  • BV, bone volume
  • CFU-f, colony forming units-fibroblastic
  • RT-PCR, reverse transcriptase-polymerase chain reaction
  • TGF, transforming growth factor
  • TUNEL, terminal dUTP nick end labelling

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Immobilisation of limbs induces local bone loss in humans1 and animals.2 Immobilised experimental animals simulate conditions of disuse of bones, such as paralysis, plaster cast application, and long term bed rest. It has been shown that bone loss in paraplegic patients1 and sciatic neurectomised mice2 is due to increased bone resorption as well as reduced bone formation. Bone formation decreased 7–14 days after immobilisation.2 The underlying mechanism(s) of rapid reduction of bone formation after limb immobilisation has not been elucidated.

p53 Expression as well as apoptosis is observed in bone marrow cells.3 Apoptosis has also been seen in osteocytes.4 We proposed that intracellular p53 has an important role in trabecular bone turnover and in bone marrow cell development after immobilisation. Specifically, we suggested that rapid reduction of trabecular bone mass, and bone formation after limb immobilisation, are closely related to facilitation of p53 signalling in bone marrow cells. In this study we investigated the effects of p53 gene status on trabecular bone turnover and bone marrow cell development after immobilisation in p53 gene knockout (p53−/−) mice and p53 wild-type (p53+/+) mice.

MATERIALS AND METHODS

Experimental design

The p53−/− mice were homozygous for the p53 gene disruption, as described previously.5 Wild-type mice of the parental wild-type inbred strain were used as p53+/+ mice. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the University of Occupational and Environmental Health. Eight week old male p53+/+ and p53−/− mice were respectively assigned to two groups matched for body weight (groups 1 and 2 for p53+/+ mice and groups 3 and 4 for p53−/− mice; n = 32 in each group) after 1 week’s acclimatisation. Mice of groups 1 (p53+/++C) and 3 (p53−/−+C) were mobilised normally as age matched controls. The right hind limbs of mice of groups 2 (p53+/++IM) and 4 (p53−/−+IM) were immobilised for 7 days in full extension with a bandage tape, as described previously.6

Histomorphometric analysis

Histomorphometric analysis was described previously.2,7 Four groups comprising five mice each were studied. Abbreviations for histomorphometric parameters were derived from the recommendations of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee,8 as follows: BV/TV, trabecular bone volume to tissue volume (%); Tb.N, trabecular number (/mm); Tb.Th, trabecular thickness (µm); BFR/BS, bone formation rate to bone surface (µm-%/day). The right tibias of five mice in each group were stained by the terminal dUTP nick end labelling (TUNEL) assay, as described previously.6 We measured the secondary spongiosa in the proximal tibia.

Evaluation of bone marrow cells

Twelve mice in each group were killed on day 7 of immobilisation/control. Six tibias in each group were used for the colony forming units-fibroblastic (CFU-f) assay, while the other six were used for the measurement of mineralised nodule formation, as described previously.2,7

Evaluation of mRNA expression in bone marrow cells

Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification

Four groups comprising five mice each were treated as described above. mRNA was isolated from the tibial bone marrow cells and PCR amplification was performed, as described previously.7 Specific PCR primers were designed from published sequences of murine p539 and β-actin.10 The PCR products were 517 bp (p53) and 632 bp (β-actin) in fragment sizes.

Statistical analysis

Results are expressed as the mean (SD). Differences between groups were examined for statistical significance using the Mann-Whitney U test. A value of p<0.05 denoted the presence of a significant difference.

RESULTS

Body weight and tibial size

Body weight increased in all four groups 7 days after the start of the experiment (data not shown), and the body weight gain was similar in these groups. No significant differences were seen in the length and diameter of the tibias among the four groups (data not shown).

Trabecular bone volume, structure, and bone formation

Table 1 shows that BV/TV in p53+/++IM was significantly reduced to about 77% of the level of p53+/++C. Tb.N in p53+/++IM was significantly decreased compared with that in p53+/++C. On the other hand, BV/TV and Tb.N did not differ between p53−/−+C and p53−/−+IM. There were no significant differences in Tb.Th among the four groups. BFR/BS was significantly lower in p53+/++IM than in p53+/++C. However, BFR/BS in p53−/−+IM was maintained at a similar level to that in p53−/−+C. There was no difference between the control groups of p53+/+ and p53−/− mice.

Table 1

Effects of p53 gene and immobilisation (IM) on the tibial bone and bone marrow cells

Bone marrow cells

Disruption of the p53 gene and immobilisation did not change the numbers of total CFU-f (data not shown). Immobilisation significantly decreased the numbers of alkaline phosphatase (AP) positive CFU-f, and mineralised nodule formation in cultured bone marrow cells from p53+/+ mice compared with control mice (table 1). However, there were no significant differences in the numbers of AP positive CFU-f and nodule formation between p53−/−+C and p53−/−+IM.

mRNA expression

Immobilisation enhanced (about 1.8 times) p53 mRNA expression in bone marrow cells from p53+/+ mice compared with the level in the control group (fig 1).

Figure 1

p53 mRNA expression in bone marrow cells. Representative data are shown. C, control; IM, immobilised group.

TUNEL assay

In p53+/++IM, 5.4 (1.3)% of osteocytes in trabecular bone were stained by the TUNEL method (fig 2A). However, in p53+/++C, p53−/−+C, and p53−/−+IM, no osteocytes were stained (fig 2B).

Figure 2

TUNEL staining of the trabecular bone of (A) p53+/+ and (B) p53−/− mice obtained at 7 days after immobilisation. Arrows indicate TUNEL positive osteocytes. Scale bar = 50 µm. Each tissue section was finally counterstained with haematoxylin and eosin. (C) Proportion of TUNEL positive bone marrow cells. Data are mean (SD) values of five mice. *p<0.05 v p53+/++C; +p<0.05 v p53+/++IM, by the Mann-Whitney U test. The values of p53−/−+C are not significantly different from those for p53+/++C and the values of p53−/−+IM are not significantly different from those for p53−/−+C.

In p53+/+ mice the frequency of TUNEL positive bone marrow cells as well as osteocytes significantly increased in the immobilised group compared with the control group (fig 2C). However, there was no significant difference in TUNEL positive bone marrow cells between p53−/−+C and p53−/−+IM.

DISCUSSION

We demonstrated the presence of TUNEL positive osteocytes in p53+/+ mice after immobilisation, but not in p53−/− mice. Previous studies reported that cell death in the femur of glucocorticoid treated rabbits, affecting osteoblasts and osteocytes, comprised up to half of the BV and was consistent with apoptosis.11 Furthermore, in this study, we showed an increased proportion of TUNEL positive bone marrow cells in p53+/+ mice after immobilisation, coincident with increased levels of p53 mRNA. These results are in agreement with the results of our previous studies showing increased p53 mRNA levels in bone marrow cells from hind limbs of p53+/+ mice after tail suspension.7 They are also similar to the report of Ke et al, which showed that p53 expression induced by an oestrogen agonist/antagonist was localised in apoptotic cells in rat bone marrow.12 Considered together, these results indicate that limb immobilisation induces p53 related apoptosis of osteocytes and bone marrow cells.

We recently reported that articular cartilage degenerates after immobilisation in p53+/+ mouse knees, but not in p53−/− knees, and that apoptotic cells are present in articular cartilage in the femur and tibia of p53+/+ mice after immobilisation.6 We previously showed that bone loss due to mechanical unloading is closely related to facilitation of intracellular p53–p21 signalling.7 On the other hand, immobilisation induces transforming growth factor (TGF)β1 in synovial fluid.13 TGFβ1 is essential for rapid p53 mediated cellular responses, which decide cellular fate.14 The TGFβ1-p53–p21 pathway may be the critical role of signal transduction in bone marrow cells after immobilisation.

Bone marrow cell culture studies showed that immobilisation of p53+/+ mice markedly reduced the number of AP positive CFU-f, and mineralised nodule formation, although there was no significant difference in total CFU-f between p53+/++C and p53+/++IM. These results suggest that immobilisation could suppress the osteogenic cells at mature stages of osteogenic differentiation in vitro. Previous studies in the hind limb elevation model also showed that skeletal unloading inhibited the proliferation and differentiation of rat osteoprogenitor cells in vitro.15 These results suggest that intracellular p53 signalling enhanced by immobilisation leads to apoptosis of osteogenic progenitors or mature osteoblasts.

In conclusion, disruption of the p53 gene preserved bone formation and trabecular bone mass.

Acknowledgments

This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan (Scientific Research B 11470315 to AS and Scientific Research B 12470313 and Scientific Research on Priority Areas B 12137210 to TN).

REFERENCES

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