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Naslin Rasheed, Xueying Wang, Qing-Tian Niu, James Yeh, Baojie Li, Atm-deficient mice: an osteoporosis model with defective osteoblast differentiation and increased osteoclastogenesis, Human Molecular Genetics, Volume 15, Issue 12, 15 June 2006, Pages 1938–1948, https://doi.org/10.1093/hmg/ddl116
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Abstract
Atm is a Ser/Thr kinase involved in DNA damage response and is required for genome integrity and stem cell renewal. Here, we report an additional role for Atm in bone remodeling. Atm −/− mice showed reduced bone mass, especially at the trabecular bones, accompanied by a decrease in bone formation rate and defective differentiation of osteoblasts, but normal numbers of osteoprogenitor cells and osteoblasts. Atm might affect osteoblast differentiation by modulating the expression of osterix, a lineage-specific transcription factor essential for osteoblast maturation, likely via the bone morphogenetic proteins pathway. Atm −/− mice also displayed a marked increase in osteoclastogenesis and bone resorption, although Atm had no cell-autonomous effect on osteoclast differentiation and resorption. Increased osteoclastogenesis could be caused by a substantial reduction in testosterone and estradiol levels in male and female mice, respectively. The steroid hormone deficiency is a result of gonad developmental defects, which led to an increase in serum gonadotrophic hormone, FSH via a feedback regulation. Overall, these results indicate that Atm deficiency leads to osteoporosis mainly as a result of hypogonadism-induced bone resorption together with compromised osteoblast differentiation, and that Atm plays a positive role in regulating expression of osteoblast-specific transcription factor, osterix.
INTRODUCTION
Ataxia-telangiectasia (A-T) is an incurable progressive neurodegenerative disorder. Ataxia usually occurs at a very early stage and A-T patients are often confined to wheelchairs before the age of 10. Most A-T patients die in their teens or early twenties ( 1 ). The disease is caused by mutations in ATM (A-T mutated) and is accompanied by other syndromes including immunodeficiency, infertility, cancer predisposition, radiosensitivity and premature aging ( 2 – 4 ). Most of these symptoms are recapitulated in Atm knockout mice ( 5 – 7 ).
The ATM gene encodes a Ser/Thr kinase, which is activated by double-stranded DNA breaks (DSBs) ( 8 , 9 ). Its main function is to modify downstream molecules such as p53 and Brca1 to regulate cell cycle progression, apoptosis and DNA repair. As a consequence, A-T cells or murine Atm −/− cells exhibit genomic instability including telomere shortening and premature senescence. Impaired DNA damage response and repair are believed to be the underlying cause for cancer predisposition, immunodeficiency and infertility in A-T patients. ATM has been implicated in various other cellular processes as well ( 10 ). For instance, ATM was reported to regulate insulin signaling and this may explain the diabetic phenotype of A-T patients ( 11 ). ATM has also been implicated in regulating oxidative stress response and A-T cells and Atm-deficient murine cells were found to accumulate reactive oxygen species (ROS) and ROS-induced damage ( 12 – 14 ). This could be attributed to the failure of Atm −/− cells to up-regulate antioxidant proteins through the transcription factor, Nrf2 ( 15 ). An aberration in oxidative stress response was found to affect hematopoietic stem cell renewal in Atm −/− mice ( 16 ). In addition, Atm deficiency was reported to accelerate aging and stem cell renewal defect of mice with telomere dysfunction (the telomerase RNA component Terc-deficient mice) ( 17 ). Yet, the exact molecular mechanism by which ATM deficiency causes these defects are not well understood.
In an effort to study the function of Atm in bone development, we analyzed bone remodeling in Atm knockout mice and their wild-type littermates. Rapid bone growth in size and mineral deposition takes place during puberty and slows down thereafter. Bone is then constantly remodeled with old bones being replaced by newly formed bones after puberty until pre-menopause ( 18 , 19 ). In both, the growth spurt during puberty and the remodeling, steroid hormones play important roles. Bone formation is a function of osteoblasts that are derived from bone marrow mesenchymal stem cells (MSCs). Osteoblast differentiation from MSCs is a multiple-step process that requires the expression of lineage-specific transcription factors such as Runx2 and Osterix (Osx), along with other more generally expressed transcription factors like Atf4 and Dlx5 ( 20 , 21 ). Results obtained from knockout mice studies indicate that both Runx2 and Osx are essential for osteoblast maturation and bone calcification and that Osx acts downstream of Runx2. Moreover, ectopic expression of either Runx2 or Osx is sufficient to induce the expression of osteoblast-specific proteins ( 22 ). Osteoblast differentiation/maturation is promoted by growth factors and cytokines, among which bone morphogenetic proteins (BMPs) and Wnt are well studied. For example, BMP2 and BMP4 are able to drastically induce the expression of Osx and promote osteoblast differentiation ( 23 ). On the other hand, bone resorption is a function of osteoclast that shares the same precursor as macrophages and is derived from bone marrow hematopoietic stem cells (HSCs). Furthermore, there exists a functional interaction between osteoblasts/osteoprecursors and osteoclasts, as the former express cytokines such as RANKL, M-CSF and OPG to regulate differentiation of the latter, which express the cognate receptors for these cytokines ( 24 , 25 ). A coordinated action of both osteoblasts and osteoclasts is critical in maintaining optimal bone density and bone mass.
Bone disorders occur when the balance between bone formation and bone resorption is disrupted. Osteoporosis is one of the most common aging-associated bone disorders and is characterized by reduced bone mass and deterioration of bone microstructures, leaving one at an increased risk of bone fractures ( 26 ). There are mainly two types: post-menopausal osteoporosis, affecting 40% of women and caused by hyperactivity of osteoclasts due to estrogen shortage; and aging-associated osteoporosis, affecting both men and women and caused by a decline in bone formation (loss of 20–30% of cancellous bones and 5–10% of cortical bones) ( 18 , 20 , 27 ). Estrogen deficiency is known to induce bone resorption. This can be mediated by osteoblasts as estrogen deficiency was found to induce expression of M-CSF and RANKL, which stimulate osteoclastogenesis. Estrogen deficiency is also believed to stimulate bone formation due to the coupling of bone resorption and bone formation, at least at the early stage of estrogen deficiency ( 18 , 19 ). But bone resorption outpaces bone formation in units (BMUs), leading to a net loss of bone and resulting in osteoporosis. We report here that Atm −/− mice show osteoporotic phenotypes, accompanied by decreased bone formation and increased bone resorption. Bone formation defect is likely caused by defective osteoblast differentiation, rather than a shortage of osteoprogenitor cells. The differentiation defect is associated with a downregulation of Osx but not Runx2 or Atf4, suggesting that reduction in osterix might mediate the defective differentiation. Furthermore, Atm deficiency compromised the induction of Osx by BMP2, which is synthesized and secreted by osteoblasts and plays critical roles in osteoblast differentiation and bone formation. The increased bone resorption is very likely caused by hypogonadism-induced osteoclastogenesis. Our data also suggest that Atm deficiency results in hypogonadism due to defective gonad development itself rather than a shortage of gonadotrophins. Therefore, Atm-deficient mice represent a model for osteoporosis with steroid shortage and decreased bone formation.
RESULTS
Atm −/− mice showed reduced bone mass
To determine whether Atm deficiency affected bone remodeling, mice deficient for Atm and their control littermates (4-months-old) were analyzed for bone mass by dual X-ray absorptiometry and bone histomorphometry. The mutant mice showed a slight reduction in bone mineral density and a 60% reduction in the trabecular bone volume (Table 1 ). The number of trabecular bones was decreased by 40% and the separation of trabecular bones was markedly increased (Table 1 ). Therefore, the mutant mice exhibit an osteoporotic phenotype at the cancellous bones rather than the cortical bones. These results suggest that the balance between bone formation and bone resorption during bone remodeling was disrupted in Atm −/− mice. We also found that both male and female Atm −/− mice showed osteoporotic phenotypes (data not shown). Histomorphometry analysis of 6-week-old mice revealed a 25% reduction in bone volume in the absence of Atm (12.6±1.7% for −/− versus 16.6±2.5% for +/+), a 20% reduction in the number of trabecular bones (4.98±1.2 for −/− versus 6.18±0.8 for +/+, #/mm). We decided to focus on 4-month-old Atm −/− mice as they showed more severe osteoporotic phenotypes and are in the phase of bone remodeling, whereas 6-week-old mice are at the stage of pubertal growth spurt that is under the control of growth hormone and insulin-like growth factor 1 (IGF-1), which decline after puberty.
Decreased bone formation in Atm −/− mice
One cause of bone loss is defective bone formation by osteoblasts. To measure the bone formation rates in Atm −/− and control mice, calcein was injected twice at an interval of 7 days into both mutant and wild-type mice, which were sacrificed 2 days after the second injection. Histomorphometric analysis of the femurs revealed decreased bone formation rates in Atm −/− mice at the trabecular bones, but not much difference was seen at the periosteal or endosteal surface of cortical bones (Table 1 and data not shown). However, no significant change was observed in osteoblast surface and the number of osteoblasts in Atm −/− mice, suggesting that the decrease in bone formation rate could be a result of compromised osteoblast differentiation/maturation, rather than the decline in the number of osteoblasts (Table 1 ), and that reduced bone formation might contribute to the osteoporotic phenotypes of Atm −/− mice.
Defective differentiation/maturation of Atm −/− osteoblasts
To test whether decreased bone formation was caused by a cell-autonomous effect of Atm on osteoblasts, we studied the differentiation potential of osteoblasts in the presence or absence of Atm. Primary calvarial osteoblasts were isolated and their differentiation was monitored by the specific markers, ALP and mineralization. As shown in Figure 1 A and B, histochemical staining of ALP and quantitative assay of ALP revealed a decrease in ALP activities in Atm −/− osteoblast cultures. We found a similar decrease in mineralization bone nodules in Atm −/− osteoblast cultures (Fig. 1 C). RT–PCR assays also showed a reduction of procollagen α1 (data not shown). These results indicate that Atm has a cell-autonomous effect on osteoblast differentiation, consistent with the reduced bone formation seen in Atm −/− mice.
Atm deficiency did not alter the number of bone marrow osteoprogenitor cells
To test whether Atm has a role in osteoblast stem cell renewal, we compared the numbers of osteoprogenitor cells in the bone marrow of Atm −/− and control mice. One difference we observed is that the colonies from Atm −/− mice were significantly smaller in size than those from the control mice, indicating that Atm −/− osteoprogenitor cells might proliferate at a reduced rate in vitro . We also observed a compromised ALP staining in Atm −/− cultures. However, careful counting of these colony forming units (upto day 10) did not reveal a significant difference in the number of ALP positive colony formation units, an indication of the number of osteoprogenitor cells (Fig. 1 D and E). More importantly, even in 8-month-old mice, Atm −/− mice showed a normal number of osteoprogenitor cells, similar to that of 4-month-old mice, indicating that aging (upto 8 months) has no significant effect on the number of osteoprogenitor cells in mice (data not shown). These results clearly show that Atm did not affect the stem cell population of osteoblast lineage during bone remodeling.
Atm deficiency down-regulated the expression of osterix
Calvarial osteoblasts deficient for Atm showed a compromised differentiation, manifested by a decrease in ALP expression and a decease in bone nodule formation in vitro (Fig. 1 ). To understand the molecular mechanisms behind the differentiation defect observed in Atm −/− osteoblasts, we tested the expression of the transcription factors that are critical for osteoblast differentiation, as well as other osteo-markers. The same number of Atm −/− and control osteoblasts were plated and cultured in differentiation medium. At day 1, 4 and 7, a plate was harvested and total RNA was isolated. These RNA samples were used to perform semi-quantitative RT–PCR assays. It was found that Atm −/− osteoblasts expressed the same levels of Runx2 and Atf4 as wild-type osteoblasts. Yet, the mutant osteoblasts showed significantly reduced levels of osterix (Fig. 2 A). Western blot analysis confirmed reduced expression of Osx at the protein level in Atm −/− osteoblasts (Fig. 2 B). As Osx is essential for osteoblast differentiation and bone calcification and that the levels of Osx determine the expression of osteoblast-specific markers, we believe that the downregulation of Osx might mediate the effects of Atm deficiency on osteoblast differentiation.
These results suggest that Atm might participate in a signaling pathway that controls Osx expression. One of the best studied pathways that regulate Osx expression is the BMP-Smad1/5/8 pathway, although it is likely that Osx is not a direct target gene of Smad1/5/8 ( 28 ). BMP2-induced up-regulation of Osx was also significantly compromised in Atm −/− osteoblasts (Fig. 2 C), suggesting an important role in the BMP-induced Osx upregulation and a possible link between the BMP-Smads pathway and the Atm signaling pathway. The data also point to an important role for Osx in sensing various stimuli and in controlling osteoblast differentiation ( 29 , 30 ).
Increased bone resorption in Atm −/− mice
We have shown that Atm −/− mice exhibited an osteoporotic phenotype, accompanied by decreased bone formation and osteoblast differentiation. Yet, the disparity between the reduction in bone mass at cancellous bones and the extent of the reduction in bone formation rate suggests that bone resorption might also be altered in Atm −/− mice at the phase of remodeling. Further histomorphometric analysis of 4-month-old mice revealed a significant increase in the bone resorption surface and in the number of osteoclasts in Atm −/− mice (Fig. 3 A and B). Moreover, Atm −/− mice showed an increase in excretion of urine deoxypyridinoline (DPD) crosslinks, a marker for in vivo bone resorption (Fig. 3 C). However, no obvious increase in bone resorption and osteoclastogenesis was observed in 6-week-old Atm −/− mice (data not shown). These data indicate that Atm −/− mice have increased bone resorption in addition to decreased bone formation during bone remodeling but not during pubertal growth spurt.
Atm did not affect osteoclast differentiation and resorption in vitro
Increased bone resorption and osteoclastogenesis could be caused by a cell-autonomous role for Atm in osteoclast lineage. Alternatively, Atm might indirectly affect osteoclastogenesis, e.g. via hormonal regulation. To distinguish the two possibilities, we compared osteoclast differentiation in Atm −/− and control bone marrow monocytes. The number of TRAP positive osteoclasts are similar for both Atm −/− and control monocyte cultures (Fig. 4 A and B), indicating that Atm does not directly affect osteoclast differentiation. Pit formation assay on dentine discs revealed no significant difference in osteoclast resorption activities between Atm −/− and control osteoclasts (Fig. 4 C). These results indicate that Atm may not have a cell-autonomous effect on osteoclast differentiation or resorption.
Osteoblasts can synthesize and secrete cytokines to regulate osteoclast differentiation from HSC in the bone marrow. Among the best studied cytokines are RANKL and M-CSF, both required for proliferation and differentiation of osteoclast, and OPG, a decoy receptor for RANKL that inhibits osteoclast differentiation ( 24 , 25 ). We then compared the expression of these cytokines in Atm −/− and control osteoblasts using semi-quantitative RT–PCR assays. No significant difference was observed for all three cytokines at mRNA levels (Fig. 4 C), suggesting that Atm deficiency might not affect the potential of osteoblasts in supporting osteoclastogenesis.
Steroid hormone shortage in Atm −/− mice
We have shown that Atm −/− mice display an osteoporotic phenotype and exhibit increased bone resorption. Yet, Atm deficiency did not affect either osteoclast differentiation from bone marrow monocytes or osteoclast resorption activity. These results suggest that Atm deficiency augments bone resorption and osteoclastogenesis without a cell-autonomous effect on osteoclasts. One of the best studied risk factors for osteoporosis is deficiency of steroid hormones, which are crucial for proper bone remodeling by inhibiting osteoclastogenesis. For example, post-menopausal women and ovariectomized mice show increased osteoclastogenesis and osteoporosis ( 31 , 32 ). Steroid hormone shortage is known to promote osteoclastogenesis, osteoclast activity and survival. One mechanism is through controlling the production of cytokines such as M-CSF and RANKL by osteoblasts. It was previously reported that Atm −/− mice are infertile due to defective gametogenesis ( 7 , 33 ). To test whether production of steroid hormones is altered in Atm −/− mice, we measured serum and urine levels of testosterone and estrogen in male and female mice, respectively. Male Atm −/− mice had markedly reduced levels of testosterone and significantly atrophied testes when compared with the control mice (Fig. 5 A–C). A marked decrease in estradiol levels and atrophied ovaries were also observed in female Atm −/− mice (Fig. 5 D–F). Because of the crucial roles for testosterone and estradiol in osteoclastogenesis and bone remodeling, the osteoporotic phenotypes of Atm −/− mice might be at least partially caused by increased osteoclastogenesis resulting from hypogonadism.
Hypogonadism can be a result of shortage of gonadotrophic hormones that are secreted by pituitary, or due to developmental defect of gonads. Previous studies indicated that Atm −/− mice are infertile due to defects in gametes maturation as Atm is required for proper DNA recombination and meiosis ( 6 , 33 , 34 ). To understand what causes hypogonadism in Atm −/− mice, we analyzed the serum levels of FSH and LH, two of the most important gonadotrophic hormones. It was found that Atm −/− mice showed a significant increase of serum FSH in both male and female (Fig. 6 A and B), suggesting that the hypogonadism is unlikely to be caused by a defect in the hypothalamus–pituitary axis. Instead, the rise in FSH reflects a feedback regulation on hypothalamus–pituitary–gonadotrophin release by steroid hormones deficiency. This feedback regulation has been observed in post-menopausal women, gonadectomized mice, as well as in mice with targeted estrogen receptors [reviewed in ( 35 , 36 )]. On the other hand, LH levels were not significantly altered based on two assays (data not shown). Similar cases have been reported in which FSH was selectively elevated by gonadectomy ( 37 , 38 ). We cannot exclude the possibility that the assays were not sensitive enough for low levels of LH in mouse serum. Nevertheless, these results overall suggest that hypogonadism of Atm −/− mice is most likely due to an autonomous effect of Atm deficiency on development of ovary and testis, rather than a defect in gonadotrophin synthesis and secretion. Moreover, the pituitary appeared morphologically normal in Atm −/− mice (data not shown), in contrast to the severely hypotrophied gonads (Fig. 5 ). This is supported by the observation that even 6-week-old mice showed upregulation of FSH and that Atm −/− mice display spermatocyte degeneration as early as postnatal day 8 and oocyte degeneration in the later stage of embryogenesis. Degeneration leads to extensive apoptotic gamete precursors and a structural alteration of ovary and testis ( 33 ).
Atm-regulated osteoblast differentiation independent of estrogen
It has been previously reported that estrogen as well as androgen can directly affect the osteoblast differentiation, although inconsistent results were obtained ( 18 , 19 ). It is believed that the variable results are due to difference in the expression of estrogen receptor isoforms, stages of differentiation and cell lines used ( 19 ). To test whether the differentiation defect of Atm −/− osteoblasts involves steroid hormone shortage, bone marrow MSCs from Atm −/− and control mice were treated with increasing amounts of estrogen and their differentiation was compared. It was found that estrogen could indeed enhance the differentiation of MSCs to osteoblasts, manifested by an increase in ALP expression (Fig. 6 C). However, estrogen, even at high concentrations, was not able to rescue the differentiation defect of Atm −/− osteoblasts to the extent of wild-type cells (Fig. 6 C). These results indicate that Atm has a cell-autonomous effect on MSC differentiation to osteoblasts independent of estrogen. This conclusion is supported by our observation that estrogen did not have a significant effect on calvarial osteoblast differentiation of either Atm −/− or control cells, with Atm −/− cells showing compromised differentiation (data not shown and Fig. 1 A–C).
DISCUSSION
The roles for Atm in DNA damage response and tumorigenesis have been extensively studied. Yet, how Atm deficiency leads to aging-associated phenotypes is not well understood. Previous studies have indicated that Atm might affect stem cell renewal and this may regulate the aging process ( 16 , 17 ). Our genetic evidence presented here indicates that Atm −/− mice show another aspect of aging-associated phenotype in osteoporosis. The bone mass showed a slight reduction at the pubertal growth stage and a severe reduction at the remodeling phase. Both our in vivo and in vitro data indicate that the osteoporotic phenotype is a result of at least two defects during bone remodeling: decreased bone formation and increased bone resorption.
How does Atm deficiency result in enhanced bone resorption and increased osteoclastogenesis in mouse? Our current results exclude a cell-autonomous role for Atm in osteoclastogenesis and osteoclast resorption, as differentiation of bone marrow monocytes to osteoclasts was not affected by Atm deficiency and osteoclasts lacking Atm showed normal resorption activity in cultures. Moreover, Atm appears to have no effect on osteoblast synthesis of M-CSF, RANKL or OPG. It is most likely that the effect of Atm on bone resorption is mediated by a steroid hormone shortage, as Atm −/− mice show hypogonadism, with reduced levels of testosterone in males, and reduced levels of estradiol in females. This is the first link between Atm deficiency and steroid hormone shortage. Estrogen shortage is known to cause osteoporosis mainly by increasing bone turnover rate, with both increased bone formation and bone resorption, and disruption of the balance of these two events, with bone resorption outstripping formation ( 18 ). At the cellular level, estrogen deficiency directly acts on osteoblasts to enhance synthesis and secretion of IL-6, IL-11, M-CSF and other cytokines, leading to increased osteoclastogenesis ( 19 ). Estrogen is also believed to directly affect osteoblast differentiation although no consensus has been reached. We found that estrogen had no significant effect on calvarial osteoblast differentiation, but showed a stimulatory effect on differentiation of bone marrow MSCs into osteoblasts. This is consistent with previous studies using primary bone marrow MSCs ( 39 ). Yet Atm −/− and control cultures displayed a similar response to estrogen, suggesting that Atm plays no role in estrogen-dependent pathways. On the basis of the well-established link between hypogonadism and the development of osteoporosis ( 18 , 31 ), we believe that osteoporotic phenotypes of Atm −/− mice can be attributed to hypogonadism, and that female Atm −/− mice can be used as an osteoporosis model for aged post-menopausal women, who suffer from both estrogen deficiency and impaired bone formation.
Our results also suggest that hypogonadism of Atm −/− mice is of peripheral origin rather than central origin. If hypogonadism is a result of defects at hypothalamus–pituitary, gonadotrophic hormones would be low. Instead, we observed an elevated FSH levels even in young mice. This is an indication of a feedback regulation of gonadotrophins by steroid hormones. Our conclusion is further supported by the observation that Atm −/− mice display gamete degeneration/apoptosis in embryogenesis or very early postnatal development ( 33 , 35 ). This study may also provide a clue to how Atm deficiency might promote the aging process. Aging is accompanied by a marked decline of steroid and other hormones, which are believed to possess anti-aging activities ( 40 , 41 ). Further studies will be necessary to test whether some of the other phenotypic features observed in Atm −/− mice could be attributed to hypogonadism.
Atm −/− mice showed reduced bone formation, with the number of osteoblasts per bone surface not altered in the bone remodeling phase. Neither is the number of osteoprogenitor cells altered in the bone marrow, suggesting that stem cell renewal for osteoblast lineage was not significantly affected in Atm −/− mice. These observations also suggest that compromised bone formation is unlikely to be caused by the depletion of osteoprogenitor cells or a lack of osteoblasts. This is further supported by our observation that the bone marrow of mice upto 8 months did not lose the ability of osteoblastogenesis.
However, calvarial osteoblast differentiation to mature osteocytes is defective in Atm −/− calvarial osteoblast cultures, suggesting that reduced bone formation in Atm −/− mice might be, at least in part, contributable to osteoblast differentiation defect. Although steroid hormone shortage has been reported to affect osteoblast differentiation, our results indicate that Atm has a cell-autonomous effect on osteoblast differentiation, independent of estrogen. First, calvarial osteoblasts, which are not responsive to steroid hormones, showed compromised differentiation in the absence of Atm. Secondly, although differentiation of bone marrow MSC into osteoblast can be promoted by estrogen, Atm −/− and control wild-type osteoblasts displayed similar response to estrogen even at very high concentrations. In other words, estrogen could not overcome the differentiation defect of Atm −/− osteoblasts. Thirdly, estrogen deficiency is generally accompanied by increased bone formation in addition to increased bone resorption [( 42 ) and reviewed in ( 18 )]. We observed the opposite, reduced bone formation. This underscores the role for Atm in osteoblast differentiation and function. These results clearly indicate that Atm has a cell-autonomous effect on osteoblast differentiation and on bone formation in vivo . This conclusion is further supported by the findings that deficiency of c-Abl or p53, both interacting proteins and downstream targets of Atm ( 43 , 44 ), leads to altered osteoblast differentiation and bone formation as well ( 29 , 45 ), without directly affecting osteoclast differentiation or function.
What are the molecular mechanisms by which Atm deficiency causes defects in osteoblast differentiation and bone formation? From the available data, we believe that down-regulation of Osx might at least partially mediate the effect. This is because Osx appears to act at the center of osteoblast differentiation. In Osx-deficient mice, no mature osteoblasts were formed and no bone calcification was observed in vivo ; up-regulation of Osx, by ectopic expression or by BMP induction, leads to elevated expression of osteo-specific markers ( 22 ). Osx has been shown to mediate the effect of p53 deficiency and Cox2 inhibition on osteoblast differentiation ( 29 , 30 ). Moreover, c-Abl deficiency leads to defective osteoblast differentiation, associated with reduced expression of Osx ( 29 ). Up to now, how expression of Osx is regulated is not well understood. Osx expression can be induced by BMP2/4 ( 23 ), or repressed by TNF1 and p53 ( 29 , 46 ). We found that Atm deficiency diminished the BMP induced up-regulation of Osx, suggesting that Atm might participate in a step(s) from BMP2-triggered signaling pathway to the activation of Osx transcription. But how Atm regulates the transcription of Osx or the BMP signaling pathway awaits further investigation. Atm can be localized in both the cytoplasm and the nucleus and exerts differential functions ( 11 , 47 ). For example, Atm was reported to regulate the expression of IGFR in the nucleus in response to DNA damage and this may contribute to cell growth and survival ( 48 , 49 ). Atm can also phosphorylate eIF-4E-binding protein 1 in the cytoplasm and regulate insulin signaling ( 20 ). The bone phenotypes and Atm −/− osteoblasts might provide a suitable system to study the crosstalk between BMP signaling pathway and Atm.
Our previous and current studies suggest that both Atm and p53 are able to regulate the expression of Osx and osteoblast differentiation ( 29 ). It is well documented that in response to DNA damage, especially DSBs, Atm acts upstream of p53 and is required for p53 phosphorylation at Ser15, leading to stabilization and/or activation of p53 ( 9 ). Here, p53 channels the Atm's effect in DNA damage response. However, these two proteins appear not to use the same pathway to regulate Osx. First, Atm deficiency leads to a downregulation of Osx, whereas p53 deficiency leads to an upregulation. Secondly, p53 was found to directly suppress Osx promoter activity, whereas Ser15 phosphorylation, a major site of Atm, does not significantly affect the suppressive activity of p53 in the same settings ( 29 ). Thirdly, there is no evidence to support a relationship between DNA damage and osteoblast differentiation. Thus, Atm and p53 appear to regulate Osx expression in different ways. This is similar to the observation that Atm −/− MEFs undergo premature senescence, whereas p53 −/− MEFs can escape senescence. It is also in line with the concept that Atm and p53 functionally interact in a more complex manner in cell proliferation and radiosensitivity ( 50 , 51 ). Their precise relationship in regulation of Osx expression awaits further investigation on mice deficient for both Atm and p53 and primary osteoblasts derived from these mice.
In summary, we defined a novel role for Atm in bone remodeling. Atm deficiency leads to an osteoporotic phenotype in mouse due to reduced bone formation and increased bone resorption. Atm controls bone formation mainly by regulating osteoblast differentiation via the transcription factor Osx. Atm controls bone resorption through its effect on gonadogenesis and synthesis/secretion of steroid hormones. These results indicate that Atm −/− mice is an osteoporosis animal model similar to that affecting post-menopausal women. Moreover, our results also support an involvement of Atm, a Ser/Thr kinase, in regulating the expression of an osteoblast-specific transcription factor, osterix.
MATERIALS AND METHODS
Mice and cell cultures
Atm −/− mice (129S6/SvEvTac- Atm tm1Awb , Jackson Laboratories) were crossed to C57BL/6 six times. Primary osteoblasts were isolated from newborn pups as previously described ( 45 ). Briefly, calvaria from new-born pups were excised and washed in PBS. They were then digested in MEM containing 0.1% collagenase type I and dispase for 10 min at 37°C and the supernatant is discarded. The calvarias were digested four more times with fresh enzymatic solutions for 15 min each at 37°C and the supernatants were pooled, which were then spinned down to collect the cells. The cells were cultured in α-MEM with 15% FCS for 1 week until confluency and then used for experiments.
In vitro osteoblast differentiation assay and CFU assay of bone marrow cells
Calvarial osteoblasts (passage 2) were used for differentiation assays as described ( 45 ). In order to limit the potential effect of cell growth on differentiation, 1.5×10 5 cells were plated into each well of 12-well plate to ensure that the cells would be confluent the following day. They were cultured in differentiation medium (αMEM medium containing 50 µg/ml ascorbic acid and 10 m m β-glycerol-phosphate) and were re-fed every 3 days. The relative ALP activity is defined as millimoles of p -nitrophenol phosphate hydrolyzed per minute per milligram of total protein (units). To assay mineral deposition, cells were cultured for 21 or 28 days. The plates were then stained for nodules with Von Kossa method. To determine the number of osteoprogenitor cells in the bone marrow, total bone marrow cells were flushed out from femurs of Atm −/− mice and their control littermates at different ages (1.5, 4 and 8 months). A total of 5×10 6 cells were plated per well of six-well plates and cultured in α-MEM with 15% FCS. After progressive periods of time in culture, the plates were stained for ALP. Colonies with more than 20 cells were counted.
Osteoclastogenesis and bone resorption assays
For osteoclast differentiation, bone marrow of 3–4-month-old mice were flushed, the monocyte fraction was isolated by centrifugation on a Ficoll plus lymphocyte separation medium gradient (ICN Biomedicals), washed and seeded at 7.5×10 4 cells/well of 96-well plates and cultured for 7 days in differentiation medium (α-MEM containing 10% FCS (Invitrogen), 30 ng/ml M-CSF (R&D Systems), and 50 ng/ml soluble recombinant RANKL (Sigma–Aldrich, St Louis, MO, USA). TRAP staining was carried out using an acid phosphatase kit (Sigma–Aldrich). Osteoclast resorption function was assessed by a pit formation assay on dentine slices (Osteosite). Monocytes were cultured for 2–3 days in the presence of 30 ng/ml M-CSF and 50 ng/ml soluble recombinant RANKL, counted and plated onto dentine slices that were pre-incubated with serum for 2 h. After 7 days, the dentine slices were sonicated in 0.5 m ammonium hydroxide and then stained with Gill haematoxylin or Toluidine blue for 2 min, washed with water and photographed under a light microscope. The resorbed areas were measured using a densitometry system and were normalized to the number of osteoclasts in the well. Urine levels of DPD cross-links were determined using commercial kits (Quidel Corporation) and were normalized to urine creatinine following the manufacturer's protocols.
Western blot analysis
Cells were lysed in TNEN buffer (50 m m Tris, 150 m m NaCl, 5 m m EDTA, 0.5% NP-40, 0.1% Triton X-100) supplemented with 1 m m NaF, Na 2 VO 3 , 1 m m PMSF, 1 µg/ml of aprotonin, leupeptin and pepstatin. Protein concentration was determined using the Bio-Rad assay. Proteins were resolved by SDS–PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Millipore). Anti-osterix was generated by injecting rabbits with a synthesized peptide (Biogenes). Anti-actin antibodies are obtained from Sigma.
RNA isolation and RT–PCR assay
Total mRNA was isolated from osteoblasts growing on 60 mm dishes using TRIzol reagent (GIBCO), subjected to DNase treatment (Ambion) and quantitated. A total of 5 µg mRNA was reverse-transcribed into cDNA using AMV (Roche Diagnostic) reverse transcriptase. The total reaction was used in the PCR to assay for the presence of osterix, Runx2, or actin with the following primers: Osterix: forward, 5′-TGA GGA AGA AGC CCA TTC AC; reverse, 5′-ACT TCT TCT CCC GGG TGT G. Runx2: forward, 5′-TGG CAG CAC GCT ATT AAA TC; reverse, 5′-TCT GCC GCT AGA ATT CAA AA. β-actin: forward, 5′-AGA TGT GGA TCA GCA AGC AG; reverse, 5′-GCG CAA GTT AGG TTT TGT CA. Atf4: forward, 5′-TTC CAC TCC AGA GCA TTC CT; reverse, 5′-CAG GTG GGT CAT AAG GTT TG. RankL: forward, 5′-CAG AAG ACA GCA CTC ACT GC; reverse, 5′-GAA CCC GAT GGG ATG C. Opg: forward, 5′-CTG CCT GGG AAG AAG ATC AG; reverse, 5′-TTG TGA AGC TGT GCA GGA AC. M-Csf: forward, 5′-CTG GAA GGA GGA TCA GCA AG; reverse, 5′-ATG TCT GAG GGT CTC GAT GG. Procollagen α1: forward, 5′-GCC TTG GAG GAA ACT TTG CTT; reverse, 5′-GCT TCC CCA TCA TCT CCA TTC. PCR was carried out for 30 cycles of denaturation (94°C/30 s), annealing (57°C/30 s), extension (72°C/1 min) and one cycle of final extension (72°C/10 min), which was just enough to detect the PCR products of osterix and Runx2.
The detection and quantification of target mRNA were performed with semi-quantitative RT–PCR. The amplification for each mRNA was performed in the linear range for RT–PCR by optimizing the template concentration and limiting the amplification cycles to below 30 to ensure exponential amplification. RT–PCR results (negative images of gels) were scanned with a Molecular Dynamics scanning densitometer. The relative levels of mRNA or protein of interest were then determined by measuring the intensity of the corresponding bands. All values were averages of cell cultures isolated from at least three Atm −/− mice and their control littermates and were normalized to the constitutive expression of the housekeeping genes.
Preparation of bone specimens
All mice were labeled with 15 mg/kg of calcein subcutaneously (Sigma Chemical Co.) twice in an interval of 7 days before sacrifice. The right femur of each animal was dissected free of soft tissue and used for measurement of femoral bone density by a dual energy X-ray absorptometer. The right tibia was dissected and cut into three equal parts. The right proximal tibia and tibial shaft were fixed in 70% ethanol solution for 2 days, and immersed in Villanueva Osteochrome Bone Stain (Polysciences Inc., Warrington, PA, USA) for 5 days. The specimens were dehydrated by sequential changes of ascending concentrations of ethanol (70, 95 and 100%) and xylene and then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester, NY, USA). Frontal sections of the proximal tibia were cut at 5 µm using a microtome (Leica RM2155, Germany) and cross-sections of the tibial shaft proximal to the tibiofibular junction were cut at 40 µm using a diamond wire Histo-Saw machine (Delaware Diamond Knives Inc., DE, USA). All sections were coverslipped with Eukitt (Calibrated Instruments Inc., Hawthorne, NY, USA) for static and dynamic histomorphometric analysis.
Bone densitometry and histomorphometric analysis
Right femoral bone mineral content (BMC) and bone mineral density (BMD) were determined utilizing a Hologic QDR-1000W dual-energy X-ray absorptiometer. The machine was adapted for an ultra-high-resolution mode with line spacing of 0.0254 cm, resolution of 0.0127 cm and a collimator of 0.9 cm diameter. The bones were placed in a Petri dish. To simulate soft tissue density surrounding the bones, tap water was poured around the bones to achieve a depth of 1 cm. Results are given for BMC and for area; area BMD is calculated as BMC/area. In addition to results for total femur, the distal and mid-region of the femur were analyzed as sub-regions. Coefficients of variation for these measurements in our laboratory are 0.8, 1.0 and 0.6%, respectively.
Histomorphometric parameters of cancellous and cortical bones in the proximal tibia and tibial shaft were measured with a digitizing morphometry system, which consists of an epifluorescent microscope (Olympus, BH-2), a color Video-Camera and a digitizing pad (Numonics 2206) coupled to an IBM computer and a morphometry program ‘OsteoMetrics’ (OsteoMetrics Inc., Atlanta, GA, USA). Measured parameters in cortical bone included total tissue area, periosteal perimeter, marrow area, endosteal perimeter, periosteal and endosteal single and double-labeled perimeters, inter-labeled widths and intra-cortical resorption area. They were then used to calculate percent cortical bone area [(total tissue area − marrow area−intra cortical resorption area)/total tissue area×100%], percent intra-cortical porosis [(intra-cortical resorption area/cortical area)×100%], periosteal and endosteal bone formation rate (BFR) [(double-labeled perimeters+single-labeled perimeters/2)×inter-labeled widths/interval time/periosteal perimeters] according to the standard nomenclature ( 52 ).
Measured parameters of cancellous bone included total tissue area, trabecular bone area and perimeter, single and double-labeled perimeters and inter-labeled widths. They were then used to calculate percent cancellous bone volume (trabecular bone area/total tissue area×100%) and cancellous BFR [(double-labeled perimeters+single-labeled perimeters/2)×inter-labeled widths/interval time/trabecular perimeters]. The region of bone measured in all groups is 1–4 m m from the growth plate in the proximal tibia. All measurements and calculations were referenced to the standard nomenclature ( 52 ).
Measurement of steroid and gonadotrophic hormones
The plasma and urine levels of testosterone and estradiol were determined by ELISA following the manufacturers' protocol (Cayman Chemicals). Samples were collected from 1.5–4-month-old Atm −/− and their control littermates. Serum levels of FSH and LH were determined by National Hormone and Peptide Program (Torrance, CA, USA) and the levels of LH was also assessed with ELISA kits from Bioserv Diagnostics.
Statistical analysis
Each experiment was repeated with three or more mutant and control mice. Statistical analysis was performed using an unpaired t -test (STATISTICA). Significant association was defined when P <0.05 (*) was compared with control.
ACKNOWLEDGEMENTS
We thank Drs Ben Li, Qiang Yu, Wai Fook Leong, Simon Cool and Tej K. Pandita for helpful discussions, Hang In Ian, Deyu Cai and Goh Choon Hong for technical support. This work was supported by the Career Enhancement Award of American Society of Bone and Mineral Research (to B. Li) and the Agency for Science, Technology and Research of the Republic of Singapore. N. Rasheed, X. Wang and B. Li are adjunct members of the Department of Medicine of National University of Singapore. Funding to pay Open Access publication charges for this article was provided by the Career Enhancement Award from ASBMR.
Conflict of Interest statement . The authors declare that they have no conflict of interest.
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
. | Atm +/+ . | Atm −/− . |
---|---|---|
Bone mineral density | 0.06±0.01 | 0.05±0.01* |
Trabecular number (#/mm) | 3.19±1.11 | 1.90±1.03* |
Trabecular separation (mcm) | 451.89±229.47 | 884.0±259.31* |
Trabecular area (%) | 13.65±3.78 | 5.40±1.89* |
Inter-label width (mcm) | 9.04±1.44 | 7.58±1.20* |
Bone formation rate/bone Pm (mcm/day) | 63.71±16.42 | 44.13±8.43* |
Peri-osteal ILW (mcm) | 6.41±1.61 | 5.48±0.55 |
Peri-osteal-MAR (mcm/day) | 0.92±0.23 | 0.78±0.08 |
Peri-osteal-BFR (mcm/day) | 43.07±21.97 | 36.73±11.87 |
Osteoblast surface/bone surface (%) | 25.05±10.28 | 23.30±7.99 |
Osteoblast number/bone surface (#/mcm) | 23.35±10.34 | 22.19±7.25 |
. | Atm +/+ . | Atm −/− . |
---|---|---|
Bone mineral density | 0.06±0.01 | 0.05±0.01* |
Trabecular number (#/mm) | 3.19±1.11 | 1.90±1.03* |
Trabecular separation (mcm) | 451.89±229.47 | 884.0±259.31* |
Trabecular area (%) | 13.65±3.78 | 5.40±1.89* |
Inter-label width (mcm) | 9.04±1.44 | 7.58±1.20* |
Bone formation rate/bone Pm (mcm/day) | 63.71±16.42 | 44.13±8.43* |
Peri-osteal ILW (mcm) | 6.41±1.61 | 5.48±0.55 |
Peri-osteal-MAR (mcm/day) | 0.92±0.23 | 0.78±0.08 |
Peri-osteal-BFR (mcm/day) | 43.07±21.97 | 36.73±11.87 |
Osteoblast surface/bone surface (%) | 25.05±10.28 | 23.30±7.99 |
Osteoblast number/bone surface (#/mcm) | 23.35±10.34 | 22.19±7.25 |
* P <0.05 ( n =8).
. | Atm +/+ . | Atm −/− . |
---|---|---|
Bone mineral density | 0.06±0.01 | 0.05±0.01* |
Trabecular number (#/mm) | 3.19±1.11 | 1.90±1.03* |
Trabecular separation (mcm) | 451.89±229.47 | 884.0±259.31* |
Trabecular area (%) | 13.65±3.78 | 5.40±1.89* |
Inter-label width (mcm) | 9.04±1.44 | 7.58±1.20* |
Bone formation rate/bone Pm (mcm/day) | 63.71±16.42 | 44.13±8.43* |
Peri-osteal ILW (mcm) | 6.41±1.61 | 5.48±0.55 |
Peri-osteal-MAR (mcm/day) | 0.92±0.23 | 0.78±0.08 |
Peri-osteal-BFR (mcm/day) | 43.07±21.97 | 36.73±11.87 |
Osteoblast surface/bone surface (%) | 25.05±10.28 | 23.30±7.99 |
Osteoblast number/bone surface (#/mcm) | 23.35±10.34 | 22.19±7.25 |
. | Atm +/+ . | Atm −/− . |
---|---|---|
Bone mineral density | 0.06±0.01 | 0.05±0.01* |
Trabecular number (#/mm) | 3.19±1.11 | 1.90±1.03* |
Trabecular separation (mcm) | 451.89±229.47 | 884.0±259.31* |
Trabecular area (%) | 13.65±3.78 | 5.40±1.89* |
Inter-label width (mcm) | 9.04±1.44 | 7.58±1.20* |
Bone formation rate/bone Pm (mcm/day) | 63.71±16.42 | 44.13±8.43* |
Peri-osteal ILW (mcm) | 6.41±1.61 | 5.48±0.55 |
Peri-osteal-MAR (mcm/day) | 0.92±0.23 | 0.78±0.08 |
Peri-osteal-BFR (mcm/day) | 43.07±21.97 | 36.73±11.87 |
Osteoblast surface/bone surface (%) | 25.05±10.28 | 23.30±7.99 |
Osteoblast number/bone surface (#/mcm) | 23.35±10.34 | 22.19±7.25 |
* P <0.05 ( n =8).
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