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Endocrinology Vol. 147, No. 5 2296-2305
Copyright © 2006 by The Endocrine Society

Runx2 Is a Target of Mechanical Unloading to Alter Osteoblastic Activity and Bone Formation in Vivo

Ruchanee Salingcarnboriboon, Kunikazu Tsuji, Toshihisa Komori, Kazuhisa Nakashima, Yoichi Ezura and Masaki Noda

Department. of Molecular Pharmacology (R.S., K.T., K.N., Y.E., M.N.), 21st Century Center of Excellence (COE) Program (K.N., Y.E., M.N.), Core to Core Program (M.N.), and Hard Tissue Genome Research Center (Y.E., M.N.), Tokyo Medical and Dental University, Tokyo 101-0062, Japan; and Department of Developmental and Reconstructive Medicine (T.K.), Division of Oral Cytology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan

Address all correspondence and requests for reprints to: Masaki Noda, Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10 Kanda-Surugadai, 2-chome, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: noda.mph{at}mri.tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Molecular mechanisms underlying unloading-induced reduction of bone formation have not yet been fully understood. In vitro, Runx2 has been suggested to be involved in mechanical signaling in osteoblasts. However, the roles of Runx2 in vivo during the bone response to mechanical stimuli have not yet been known. The purpose of this paper was to examine the roles of Runx2 in unloading-induced bone loss in vivo. Tail suspension was conducted for 2 wk using 9- to 11-wk-old Runx2 heterozygous knockout mice (Runx2+/–) and wild-type (Wt) littermates. Bones were subjected to two-dimensional micro-x-ray computed tomography, bone histomorphometry and RT-PCR analyses. Loss of half Runx2 gene dosage-exacerbated unloading-induced bone loss in trabecular and cortical envelopes. Unloading-induced reduction in mineral apposition rate and bone formation rate in cortical bone as well as trabecular bone was exacerbated in Runx2+/– mice, compared with Wt mice. Bone resorption parameters were not significantly affected by unloading or Runx2+/– genotype. Basal Runx2 and osterix mRNA levels in bone were reduced by 50% in Wt, whereas unloading in Runx2+/– mice did not further alter Runx2 and osterix mRNA levels. In contrast, osteocalcin mRNA levels were reduced by unloading, regardless of Runx2 gene dosage. These data demonstrated that full Runx2 gene dosage is required for maintaining normal function of osteoblasts in mechanical unloading or nonphysiological condition. Finally, we propose Runx2 as a critical target gene in unloading to alter osteoblastic activity and bone formation in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MECHANICAL STRESS IS an important signal to control bone remodeling. Remodeling maintains proper bone morphology and functions under the influence of mechanical stress. Mechanical stress causes deformation of bone, which initiates mechanotransduction pathway (1, 2). Presence of physical force promotes bone formation, whereas lack of physical stimuli results in loss of bone as seen in the case of disuse osteopenia or osteoporosis (3). However, the molecular mechanisms underlying mechanical stress-mediated regulation of bone cells function have not yet been fully elucidated.

Osteoblasts are bone-forming cells that are believed to be responsive to mechanical stress. Mechanical stress initiates intracellular events similar to those observed in the cases of growth factor and hormone treatment in osteoblastic cells. These events include up-regulation of the levels of cAMP, prostaglandins, and nitric oxide (4, 5, 6, 7, 8, 9). Activation of MAPK pathways, nuclear factor-{kappa}B (NF-{kappa}B) pathways, and ion channels has also been reported to transduce mechanical signals in several cell types (10, 11, 12, 13, 14). However, little is known regarding the mechanisms that link these phenomena to osteoblast-specific events in nuclei that eventually lead to the regulation of osteogenesis by mechanical stress.

One of the most important regulators of osteoblastic differentiation is Runx2 (Cbfa1), a member of runt homology domain transcription factor family. Runx2 binds to osteoblast-specific cis-acting element 2, which is located in the promoter region of osteocalcin gene (15). Expression of osteoblast phenotype-related genes such as osteocalcin, type I collagen, alkaline phosphatase, bone sialoprotein, osteopontin, and collagenase-3 is down-regulated in the absence of Runx2 gene (16).

Runx2–/– mice exhibit complete absence of osteoblasts and are embryonic lethal. However, heterozygous Runx2 knockout mice are viable and show abnormalities reported as cleidocranial dysplasia (17, 18, 19). In vitro, Runx2 mediates response of preosteoblasts to signals from the extracellular matrix via the MAPK pathway (20). However, in vivo, the roles of Runx2 in the molecular mechanisms underlying osteogenesis and mechanical stress have not yet been known.

The purpose of this study was to examine in vivo roles of Runx2 in mechanical stress-mediated bone remodeling using Runx2 heterozygous knockout mice subjected to the hind-limb unloading (tail suspension).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wild-type and Runx2+/– male mice were generated as described previously (18). Littermate mating was carried out to generate Runx2 heterozygous mutants. Genotypes were identified based on genomic PCR using DNA prepared from tail. 9- to 12-wk-old male Runx2+/– and wild-type littermate mice (24 mice in total), 18–23 g in body weight, were randomly assigned in equal numbers to loaded control and unloaded (tail suspension) groups. The mice were housed under controlled conditions at 24 C on a 12-h light, 12-h dark cycle and were fed with standard laboratory chow and given tap water. All animal experiments were approved by the animal welfare committee of our institute.

Tail suspension (hind limb unloading)
Tail suspension was conducted as follows. Briefly, a tape was applied to the surface of the tail to set a metal clip. The end of the clip was fixed to an overhead bar and the height of the bar was adjusted to maintain the mice at 30 degrees head-down tilt with the hind limbs to be unloaded. The mice in unloaded group were subjected to tail suspension for 2 wk (n = 6–8/group). Loaded control (normal housing) mice were also housed individually under the same condition except for tail suspension for the same time period (2 wk). The mice were injected ip with calcein at 4 mg/kg 9 and 4 d before they were killed at 2 wk. After 2 wk of tail suspension, mice were anesthetized with pentobarbital and killed by cervical dislocation.

Measurement of bone mineral density (BMD)
BMD of the whole femora was measured based on a dual-energy x-ray absorptiometry using PIXI apparatus (GE Lunar, Madison, WI). Ex vivo coefficients of variation were 0.5.

Micro-x-ray computed tomography (µCT) analysis
The bones were subjected to µCT analysis, using Musashi (Nittetsu-ELEX, Osaka, Japan). The data were subsequently quantified by using a Luzex-F automated image analysis system (Nireco, Tokyo, Japan). For cancellous bone, µCT images were made within the midsagittal planes in the metaphyseal region of the bones. The fractional bone volume (BV/TV) was measured in the area of 1.96 mm2 (1.4 x 1.4 mm2) with its closest and furthest edges at 0.14 and 1.54 mm distal to the growth plate of the proximal ends of the tibiae. To quantify cortical bone, µCT images of the cross-section in the middiaphyses of the femora were obtained to determine cortical bone mass. Cortical bone parameters such as cortical area and cortical thickness were analyzed.

Histomorphometric analysis of bone
For undecalcified section, femora were fixed in 70% ethanol, prestained with Villanueva osteochrome (Bone Stain), and embedded in methylmethacrylate. Cross-sections (serial 10 µm thick sections) in middiaphysis and longitudinal sections (serial 3 µm thick sections) were made using a microtome. The sections were used to examine cortical and cancellous bone formation rate (BFR) and mineral apposition rate (MAR) in periosteal, endosteal, and trabecular regions. For decalcified sections, tibiae were fixed in 4% paraformaldehyde in PBS, decalcified in EDTA, embedded in paraffin, and serial 5-mm-thick longitudinal sections made. The sections were stained for tartrate-resistant acid phosphatase (TRAP) activity. TRAP-positive multinucleated cells attached to bone were scored as osteoclasts. Measurements were made within an area in the proximal ends of the tibiae to obtain osteoclast number per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS) as defined by Parfitt et al. (21).

Deoxypyridinoline (Dpyd) measurement
Urinary Dpyd levels on d 14 of the tail suspension were measured by ELISA [Metra Biosystems, Mountain View, CA (22)]. Urine was collected from the mice in a metabolic cage during the last 24 h (on d 14 of tail suspension), and six independent samples from each group were analyzed. For these metabolic cage experiments, the mice were transferred to a new individual cage 24 h before termination of the experiments.

Semiquantitative RT-PCR analysis
For RT-PCR analysis, left femora were collected after bone marrow was flushed out. These bones were used to extract total RNA according to the acid guanidine isothiocyanate-phenol/chloroform method. RT-PCR analysis was performed using primers specific to Runx2, osterix, osteocalcin (OC), osteopontin (OPN), alkaline phosphatase (ALP), dentin matrix protein (DMP), receptor activator of NF-{kappa}B (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) genes. Reverse transcription (RT) was carried out using 1 µg total RNA, 0.2 µg oligo(dT) primer, deoxynucleotide triphosphate mix (10 mM), and Muloney murine leukemia virus-RT (100 U) in a volume of 20 µl. RNA and primer were incubated together at 65 C for 10 min and then cooled rapidly on ice before addition of other reagents. RT mixture was incubated at 37 C for 1 h. One microliter of complementary DNA was amplified by PCR in a 25-µl reaction volume containing 2.5 mM deoxynucleotide triphosphate mix, 10 µM specific primers, and rTaq DNA polymerase (1 U). After an initial denaturation at 94 C for 2 min in a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, CA), amplifications were performed at 94 C for 40 sec, 60 C for 1 min, and 72 C for 1 min. Subsequently the amplification cycle was repeated 26–32 times. The cycle number was determined so that the PCR product levels were within a linear range. Ethidium bromide-stained DNA bands were quantitated using an image analyzer Bio-1D system (Vilber Lourmat, France). As a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were also estimated by RT-PCR at 25 cycles. The ratios of the levels of mRNA expression of genes of interest relative to GAPDH expression levels were calculated.

Oligomer sets used for Runx2/Cbfa1, osterix, OC, ALP, OPN, DMP, RANK, RANKL, OPG, and GAPDH are shown in Table 1Go.


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  		TABLE 1. RT-PCR primer sequences

 
Statistical evaluations
The data were presented as mean values ± SD. Statistical analysis was conducted according to ANOVA test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Runx2+/– mice maintain bone mass in loaded condition but not unloaded condition in vivo
To examine the roles of Runx2 gene in bone mass determination under mechanical stress, 9- to 12-wk-old Runx2+/– (Het) and wild-type (Wt) littermate mice were subjected to hind limb unloading. Unloading reduced trabecular bone mass in the tibia of Wt mice as reported previously. The basal levels of trabecular bone mass in Wt and Runx2+/– mice were not significantly different, although Runx2+/– mice tended to exhibit less values than those of Wt (Fig. 1Go, A and B). However, loss of half Runx2 gene dosage (Runx2+/–) further accelerated the degree of unloading-induced trabecular bone loss (Fig. 1Go, B and D). Quantification of the fractional BV/TV indicated about 22% reduction in BV/TV in Wt mice by unloading (Fig. 1DGo, Wt), whereas in Runx2+/– mice, unloading induced about 37% reduction in BV/TV (Fig. 1DGo, Het). Thus, unloading-induced bone loss rate was increased by about 60% in Runx2+/– mice, compared with Wt (from 22 to 37%). Corresponding to BV/TV, unloading also decreased BMD in Wt by 13% (Fig. 1Go, C and E), and this was also exacerbated by about 30% in Runx2+/– mice (Fig. 1EGo, 17% reduction ratio in Het).


Figure 1
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  		FIG. 1. µCT images, trabecular BV/TV, and BMD of bone in loaded and unloaded mice. A, Midsagittal µCT images of the proximal ends of the tibiae after 2 wk of loading (L) or unloading (UL) in Wt or Runx2+/– (Ht) mice. B, Fractional trabecular BV/TV of the tibiae after 2 wk of either loading (load) or unloading (unload) in Wt or Runx2+/– (Het) mice shown in A. Analyses were conducted in the proximal ends of the tibiae as described in Materials and Methods. C, BMD of the whole femora measured by dual-energy x-ray absorptiometry. D and E, Percent reduction rates of tibial BV/TV (D) and femoral BMD (E) due to unloading, respectively. Each of the four groups consisted of six mice. Data are expressed as means and SD. Asterisks indicate statistically significant difference (*, P < 0.05; **, P < 0.01).

 
This was not limited to cancellous bone because unloading reduced cortical bone thickness in Wt mice, and loss of half Runx2 gene dosage also exacerbated such cortical bone loss (Fig. 2Go, A–C). The degree of unloading-induced reduction in cortical bone thickness [percent reduction = (loaded-unloaded)/loaded x 100] at the midshaft was about 40% greater in Runx2+/– mice, compared with that in Wt mice (Fig. 2CGo, 17 vs. 12%, respectively). Unloading-induced decrease in cortical area (cortical area shown in red in Fig. 1DGo, right panel) was significantly more in unloaded Runx2+/– mice, compared with unloaded Wt mice (Fig. 2Go, D and E). Bone area and periosteal circumference were not significantly altered by unloading in both Runx2+/– and Wt (Fig. 2Go, F and G). Runx2+/– mice also revealed more loss of cortical thickness at the distal one fourth of the total length of bone (data not shown). These data point to the role of Runx2 in vivo in the maintenance of bone mass under unloaded condition.


Figure 2
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  		FIG. 2. Cross-sectional µCT images and parameters in cortical bone. A, Cross-sectional µCT images of middiaphyses of the femora. The µCT images were obtained in a plane perpendicular to the long axis of the femoral. B, Quantified cortical thickness at middiaphyses of femora was estimated by averaging the thickness measured at eight points at every 45-degree direction. C, Percent reduction rates of cortical thickness shown in B. D and E, Cortical area at middiaphysis of femora and percent reduction rate. F and G, Periosteal circumference and bone area at middiaphyses of femora. The images were quantified by using a Luzex-F automated image analysis system. Each of the four groups consisted of six mice. Asterisks indicate statistically significant difference (*, P < 0.05; **, P < 0.01).

 
Runx2+/– mice maintain bone formation in loaded condition but not unloaded condition
Because Runx2+/– mice could maintain near-normal bone mass under loaded condition but not unloaded condition, we addressed whether this observation would be due to the Runx2 function in osteoblasts in vivo. In loaded mice, basal levels of endosteal and periosteal BFR and MAR in midshaft of the femora were not significantly different, regardless of the genotypes (Fig. 3Go, B–E). In Wt mice, unloading decreased endosteal and periosteal MAR and BFR levels by 40–80%. Surprisingly, endosteal and periosteal MAR and BFR levels were totally reduced to nearly undetectable levels by unloading in Runx2+/– mice (Fig. 3Go, B–E). In all of these bone formation parameters, unloading-induced reduction in Runx2+/– was more than those in Wt.


Figure 3
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  		FIG. 3. Calcein double labeling in cortical bone envelope. A, The calcein-labeled surface in the middiaphyseal regions of the femora after 2 wk of either loading (L) or unloading (UL) in Wt or Runx2+/– (Ht) mice. The lines of calcein labeling are shown in light green. Calcein was injected 9 and 4 d before animals were killed. The inset pictures indicate magnification of the labeling surface in the endosteal site of each photograph. B, Endosteal MAR. C, Endosteal BFR. D, Periosteal MAR. E, Periosteal BFR. Note that MAR and BFR levels of unloaded Runx2+/– mice could not be measured because there was no detectable calcein deposition observed as shown in Fig. 3AGo (Ht-UL). Asterisks indicate statistically significant difference (*, P < 0.05; **, P < 0.01).

 
In contrast to cortical bone envelope, examination in cancellous bone envelope in the metaphyses of femora of loaded Runx2+/– mice exhibited lower basal levels of MAR and BFR, compared with those of loaded Wt (Fig. 4Go, A–D). Unloading reduced MAR and BFR levels by about 30% in Wt (Fig. 4Go, E and F). Similar to the observations in cortical bone envelope, Runx2+/– mice exhibited exacerbation in unloading-induced decrease in that the reduction rate in MAR was about 50% more, compared with Wt (Fig. 4EGo, reduction rate, 30% in Wt vs. 45% in Runx2+/–), and in BFR about 130% more, compared with Wt (Fig. 4FGo, reduction ratio, 30% in Wt vs. 70% in Runx2+/–).


Figure 4
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  		FIG. 4. Calcein double-labeling in trabecular bone envelope. A, Calcein-labeled surfaces of the midsagittal planes in the distal ends of the femora. B, Magnification of calcein double-labeled surface in A. The lines of calcein labeling are indicated as light green. Calcein was injected 9 and 4 d before animals were killed. Trabecular MAR (C) and BFR (D) were measured within an area corresponding to the area used for measuring BV/TV. E and F, Percent reduction rates of trabecular MAR and BFR, respectively. Data are expressed as means and SD based on the analyses of bones from six mice per group. Asterisks indicate statistically significant difference (*, P < 0.05; **, P < 0.01).

 
Bone resorption parameters were not significantly different between Runx2+/– and Wt mice
Bone loss could be caused by reduction in bone formation, enhancement in bone resorption, or both. To examine the effects of Runx2+/– mutation on bone resorption parameters in unloading-induced bone loss, the levels of TRAP-positive cell (osteoclasts) number in vivo were examined based on histomorphometry (Fig. 5AGo). Unloading tended to marginally (not statistically significant) increase the levels of Oc.N./BS or Oc.S./BS in Wt mice, and this trend was similar in Runx2+/– mice (Fig. 5Go, B and C). To address systemic parameters of bone resorption, we also evaluated a biochemical marker of bone resorption in urine (22). Dpyd excretion into urine (measured only on the last day of unloading, d14) was not significantly altered by unloading (although it was marginally increased without statistical significance), compared with the loaded group in both Wt and Runx2+/– mice (Fig. 5DGo).


Figure 5
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  		FIG. 5. Bone resorption parameters. A, TRAP-positive cells in the decalcified sections in the proximal ends of the tibiae. Oc.N./BS (B) and Oc.S/BS (C) were measured within an area distal to the growth plate corresponding to the area of measurement for BV/TV. Osteoclast number per bone surface was calculated as the number of osteoclasts per total bone surface, and the Oc.S/BS was calculated as the percentage of bone surface covered by osteoclasts per total bone surface. Data are expressed as means and SD based on the analyses of bones from five mice per group. D, Urinary Dpyd levels. Urine samples of either loaded (load) or unloaded (unload) group from both Wt and Runx2+/– (Het) mice were collected during the last 24 h of the 2-wk tail suspension (on d 14). Urine from six to eight independent samples per group was analyzed by ELISA.

 
We next examined some of the osteoclast-related gene expression by using RNA from the cortical bone envelope. There was no major change in RANKL expression after tail suspension in Wt. However, slight up-regulation of RANKL was observed in unloaded Runx2+/– (Fig. 6GGo). Runx2+/– mice expressed only half-levels of OPG mRNA, compared with Wt, and this level was not altered by unloading (Fig. 6IGo). Interestingly, unloading up-regulated RANK gene expression in Wt but not Runx2+/– mice (Fig. 6HGo).


Figure 6
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  		FIG. 6. Semiquantitative RT-PCR analysis. Total RNA was obtained from femoral cortical bone of Wt or Runx2+/– (Het) mice subjected to 2 wk of loading (load) or unloading (unload) in. RT-PCR analyses were conducted as described in Materials and Methods, using primer specific for osteoblast- and osteoclast-related genes. A, Runx2. B, Osterix. C, OC. D, ALP. E, OPN. F, DMP. G, RANKL. H, RANK. I, OPG. The levels of each mRNA were calculated as ratios relative to GAPDH expression levels and expressed as means and SD. Samples were taken form bones of four mice in each group. Asterisks indicate statistically significant difference (*, P < 0.05; **, P < 0.01).

 
Mechanisms underlying the unloading-induced bone loss in Runx2+/– mice located downstream to Runx2 and osterix gene expression
To address the molecular profile underlying the effect of Runx2+/– mutation on unloading-induced impairment in osteoblastic function, mRNA expression levels of Runx2 gene as well as those of downstream osteoblast-related genes were examined. Basal Runx2 mRNA levels in loaded Runx2+/– mice were half of loaded Wt mice, as expected. Unloading reduced the Runx2 mRNA levels in Wt mice by about 60% (Fig. 6AGo). In contrast to Wt, unloading did not further decrease the levels of Runx2 mRNA in Runx2+/– mice (Fig. 6AGo). These observations suggest the presence of unidentified mechanisms downstream to Runx2, which could be involved in unloading-induced exacerbation of reduction of MAR and BFR in these mice.

In loaded Runx2+/– mice, osterix mRNA levels were reduced by 50% of the loaded Wt. This was compatible with the previous notion that osterix is downstream to Runx2 (23). Unloading reduced osterix mRNA level by about 60% in Wt mice (Fig. 6BGo). In contrast, unloading did not alter osterix mRNA levels in Runx2+/– mice (Fig. 6BGo). It is interesting that half-expression levels of Runx2 and osterix in loaded Runx2+/– mice did not result in major reduction in bone mass and MAR/BFR, compared with loaded Wt mice (although there was an insignificant trend). Thus, loading could compensate the loss of half-Runx2 gene dosage at least to a certain extent at the level of bone mass.

Notably, similar to bone mass, cortical MAR and BFR, the basal mRNA levels of osteocalcin, which is downstream to Runx2, were not significantly different between loaded Runx2+/– mice and Wt mice. However, in contrast to bone mass, MAR, and BFR, unloading reduced OC mRNA levels similarly in Wt and Runx2+/– mice (Fig. 6CGo). No significant change in ALP mRNA expression was observed in either the loaded or unloaded group (Fig. 6DGo). We next observed the expression pattern of DMP, another candidate gene whose expression has been shown to be regulated by Runx2 (24). Runx2+/– mice expressed similar levels of DMP mRNA, compared with Wt. Unloading significantly reduced the expression levels of DMP mRNA only in Runx2+/– but not Wt (Fig. 6FGo). Surprisingly, Runx2+/– mice expressed significantly higher levels of OPN mRNA, compared with Wt, although this level was not significantly altered by unloading. Taken together, these data suggest that the mechanism underlying unloading-induced bone loss in Runx2+/– mice could be located downstream to Runx2 and osterix gene expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we presented the first in vivo evidence that full Runx2 gene dosage is required to limit severe loss in bone mass in the absence of mechanical stimuli. Unloading by tail suspension results in substantial loss of both cortical and trabecular bones. Runx2+/– mutation exacerbated the unloading-induced bone loss. In vivo cell activity analysis indicated that Runx2+/– mutation targets unloading-induced impairment in bone formation parameters in vivo such as MAR and BFR. MAR in unloaded Runx2+/– mice demonstrated that function of individual osteoblast was significantly abolished due to Runx2+/– mutation. It has been reported that loss of half-Runx2 gene does not affect bone homeostasis in normal physiological condition (18, 19). However, in this study, we demonstrated that full dosage of Runx2 is necessary for maintaining normal function of osteoblasts in the light of mechanical unloading.

Because loaded Runx2+/– mice exhibited similar levels of BV/TV, compared with loaded Wt mice, even half-dosage of Runx2 could maintain trabecular bone volume in Runx2+/– mice to the level comparable to those of Wt. However, half gene dosage of Runx2 is not enough for the maintenance of bone mass in unloaded condition, suggesting that loading maintains bone mass at least in part through the presence of Runx2.

Bone responses to mechanical stimuli require some forms of cellular mechanotransduction. Osteocytes and bone-lining cells make up more than 95% of all cells of the osteoblastic lineage that attached to bone (25). These cells are believed to act as sensors, which are responsive to mechanical stimuli in vivo (26, 27, 28). Apparently, Runx2+/– cortex contains comparable number of osteocytes and canaliculi with that of Wt (data not shown). These observations indicated that Runx2+/– mice exhibit close-to-normal bone structure. Thus, the significant loss of cortical thickness induced by mechanical unloading in Runx2+/– mice was likely due to cell function rather than structural context.

Expression of Runx2 and osterix mRNA was down-regulated by mechanical unloading in Wt mice, showing for the first time that Runx2 and osterix are responsive to unloading in vivo. In loaded Runx2+/– mice, the Runx2 and osterix mRNA levels were about half of those in Wt, whereas such reduction appeared to be compensated during the growth in the long term to give similar levels of bone mass. Surprisingly, Runx2 and osterix levels were no longer altered by unloading in Runx2+/– mice. These data suggest the presence of mechanisms downstream to Runx2 to be involved in unloading-induced further suppression of bone formation in these mice. Because the expression of OC was consistently down-regulated in both unloaded Wt and Runx2+/– mice, such a pathway could be independent or even upstream to OC expression. Whether mechanical unloading alters posttranslational modification of Runx2 is still to be elucidated.

OPN is required for unloading-induced bone loss through as-yet-unidentified mechanisms on the side of both reduction in bone formation and increase in bone resorption. The higher OPN expression levels in the bone of loaded RUNX 2+/– mice suggest that RUNX 2 may negatively regulate OPN expression. This might happen if RUNX 2 negatively regulates OPN promoter through its binding site, but at this point we do not have any functional evidence for this notion. We do not know whether there is a direct link between the observations described in this paper and the function of OPN. Whether enhanced OPN levels in Runx2+/– mice are related to the exacerbation of unloading-induced bone loss in these mice is to be elucidated further.

Bone formation was inhibited completely by unloading in the cortical bone envelope but not so much in the cancellous bone when overall bone area and density of the bones in loaded mice are not statistically different in the Wt and RUNX 2+/– mice. We do not know whether there are differences in expression levels of Runx2 in cancellous vs. cortical osteoblasts. Another possibility is that this may be due to some compensation after long-term growth (9–12 wks) or to detection sensitivity. Although not statistically significant, unloading tended to reduce cortical bone area, cancellous bone BV/TV, and BMD. Thus, the observation that heterozygous Runx2 knockout mice exhibit further reduction in bone formation in both cortical and cancellous bone is similar in both cortical and cancellous envelopes. Because tail suspension experiments were conducted for 2 wk, the resulting difference in the degree of loss of cortical bone mass may not seem to be more than that of cancellous bone mass within this time period.

In conclusion, our data demonstrated that loss of half-Runx2 gene dosage exacerbates unloading-induced bone loss, at least in part, through the further reduction in osteoblast functions. Therefore, we propose that Runx2 is a crucial player in mechanical stress-mediated regulation in bone metabolism in vivo.


   Footnotes
 
This research was supported by the grants-in-aid received from the Japanese Ministry of Education (21st Century Center of Excellence Program, Frontier Research for Molecular Destruction and Reconstitution of Tooth and Bone, 14207056, 16659405, 16027215, 16022221), grants from Japan Space forum, NASDA, and Japan Society for Promotion of Science (Core to Core Program, Research for the Future Program, Genome Science), and Japan Society for Promotion of Science Research Grant 03142 for foreign postdoctoral fellows.

All authors have nothing to declare.

First Published Online February 2, 2006

Abbreviations: ALP, Alkaline phosphatase; BFR, bone formation rate; BMD, bone mineral density; BV/TV, bone volume; µCT, micro-x-ray computed tomography; DMP, dentin matrix protein; Dpyd, deoxypyridinoline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Het, Runx2+/–; MAR, mineral apposition rate; NF-{kappa}B, nuclear factor-{kappa}B; N.Oc/BS, osteoclast number per bone surface; OC, osteocalcin; Oc.S/BS, osteoclast surface per bone surface; OPG, osteoprotegerin; OPN, osteopontin; RANK, receptor activator of NF-{kappa}B; RANKL, RANK ligand; RT, reverse transcription; TRAP, tartrate-resistant acid phosphatase; Wt, wild type.

Received August 10, 2005.

Accepted for publication January 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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