Loss of Runx2 in Gli1 + osteogenic progenitors prevents bone loss following ovariectomy

Lay Summary

Osteoporosis weakens bones, making them fragile by disrupting the balance between cells that build bone and cells that break it down. Special cells called osteogenic progenitors help maintain healthy bones by creating new bone tissue. This study examined the role of a gene called Runx2, which is important for bone formation, in adult bone health. Using a mouse model that mimics osteoporosis after menopause, we found that removing this gene in the osteogenic progenitors prevents bone loss by reducing bone breakdown. These findings suggest that targeting Runx2 could strengthen bones and may lower the risk of osteoporosis.

osteogenic progenitors, Runx2, Gli1, osteoporosis, bone homeostasis

Introduction

Osteoporosis is an extremely common metabolic bone disorder characterized by loss of bone mass and consequent weakening of bone tissue as people age. It is the result of an imbalance between the bone-building activity of osteoblasts and bone-resorbing activity of osteoclasts. Osteoporosis is a common cause of fracture in adults, particularly in women who suffer an accelerated loss of bone in association with the estrogen deficiency of menopause.¹ Despite the availability of a number of treatments, osteoporosis remains prevalent and in need of additional therapeutic targets.²

Gli1⁺ cells are an osteogenic progenitor population critical to maintaining bone tissue homeostasis.³^,⁴ These cells give rise to osteoprogenitors and are present at the osteogenic fronts of long bones in adult mice. However, the role of Gli1⁺ cells in regulating adult bone homeostasis is not fully understood. Transcription factors have been reported to be key regulators for mesenchymal cell fate determination. Runt-related transcription factor 2 (encoded by Runx2, also known as Cbfa1),⁵ expressed by a subpopulation of Gli1⁺ osteogenic progenitors and progeny, is important in osteoblastic differentiation and maturation during embryonic development.⁴^,⁶ At that stage, Runx2 is required for cell cycle exit of preosteoblasts,⁷ late-stage maturation of osteoblasts,⁸ and ossification of developing bone. Loss of Runx2 during embryonic development prevents maturation of osteoblasts and results in a complete lack of bone formation.⁶ However, the role of Runx2 in regulating adult bone tissue homeostasis is less clear.

Here we investigated the effects of Runx2 disruption on adult long bone tissue homeostasis in mice following ovariectomy, a model for postmenopausal osteoporosis. We examined multiple parameters of bone mass and bone mineralization in sham-operated and ovariectomized Runx2 control mice (Runx2^fl/fl) and sham-operated and ovariectomized Runx2 mutant mice (Gli1Cre^ERT2;Runx2^fl/fl). Operations were performed at 2 mo of age, and bone parameters were assessed 1 and 4 mo later. Control mice exhibited significant loss of bone mass indicative of osteoporosis as early as 1 mo following ovariectomy. Deletion of Runx2 in Gli1⁺ progeny protected mice from reductions in bone mass and bone mineralization after ovariectomy, either reversing or partially mitigating bone loss associated with ovariectomy. Additionally, deletion of Runx2 resulted in reduced osteoclast activity. Our study demonstrated a preservation of bone mass following the loss of Runx2 in the Gli1⁺ cell lineage, suggesting loss of Runx2 in osteoprogenitors protects against ovariectomy-induced osteoporosis in adult mice.

Materials and methods

Generation of transgenic mice

Gli1Cre^ERT2 (JAX#00791348), tdTomato (JAX#00790549), and Runx2^fl/fl (JAX#029512) mouse lines were obtained from Jackson Laboratory. Only female mice were used for experiments involving ovariectomy.

All mice were housed in pathogen-free conditions and analyzed in a mixed background. Ear tags were used to identify mice and treatment groups. Tissue from ear biopsies was lysed by incubation in Direct PCR tail solution (Viagen 102-T) at 55 °C overnight followed by 30 min of heat inactivation at 85 °C. PCR-based genotyping was used to identify the conditional knockout and littermate control mice (GoTaq Green Master Mix, Promega, and C1000 Touch Cycler, Bio-Rad). Mice were euthanized prior to tissue harvest via carbon dioxide overdose followed by cervical dislocation. All studies were performed with the approval of the Institutional Animal Care and Use Committee of the University of Southern California. For induction of Cre lines, tamoxifen (Sigma T5648) was suspended in corn oil (Sigma C8267) at 20 mg·mL⁻¹ and injected intraperitoneally at a dose of 1.5 mg per 10 g body weight for 3 consecutive days.

For the experiment shown in Figure 1, Gli1Cre^ERT2;tdTomato mice were induced with tamoxifen at 2 mo of age and collected at 3 mo of age. Control and Gli1Cre^ERT2;Runx2^fl/fl mice were induced with tamoxifen at 1 mo of age and collected at 6 mo of age.

Figure 1

Runx2 expression by Gli1⁺ cell derivatives at the osteogenic front in adult mouse femurs. (A, B) Expression of Runx2 by Gli1⁺ progeny in the adult mouse femur. The box in A is magnified in B. The box in B is enlarged at the bottom left corner and shows co-localization between Runx2 and tdTomato. (C, D) Single channel images of panel A. (E-H) Loss of Runx2 in Gli1⁺ cells results in an increased width of cortical bone in adult mouse femurs. Boxes in E and G are magnified in F and H, respectively. Arrowheads in F and H indicate the width of cortical bone. Scale bars: 200 μm in A-D; 3 mm in E and G; 1 mm in F and H. Schematic illustrations at the bottom indicate the time points of sample collection.

For the experiments shown in Figures 2-5, Runx2^fl/fl mice received either a sham operation or an ovariectomy at 2 mo of age, and Gli1Cre^ERT2;Runx2^fl/fl mice received a sham operation or an ovariectomy at 2 mo of age. Runx2 functional changes were induced with tamoxifen 1 wk after surgery and femoral bone samples were collected at the indicated time points.

Figure 2

Runx2 deletion leads to increased cortical width in femurs. (A-H) H&E staining and microCT analysis of femurs across 4 groups of mice at 1mpt and 4mpt. Boxes in A-H are magnified in A′-H′, respectively. Representative microCT images are shown in A″-H″. Dashed lines in A′-H′ outline the cortical bone. Scale bars: 250 μm in A-H and A′-H′; 1 mm in A″-H″. (I) Statistical analysis of cortical width measured on microCT images 2 mm proximal to the epiphyseal plate of the femurs 1 and 4 mo after tamoxifen induction. Data are mean ± SD. ^* indicates p<.05; ^** indicates p<.01. Schematic illustration at the bottom indicates the time point of sample collection.

Surgical procedure

All operations were performed at 2 mo of age. Runx2^fl/fl mice were randomly assigned to receive either a sham-operation or an ovariectomy. Gli1Cre^ERT2;Runx2^fl/fl mice underwent either a sham operation or ovariectomies. Investigators were not blinded during allocation or animal handling. Mice were anesthetized with 1%-4% isoflurane (VetOne #502017). Analgesic (Buprenorphine XR, MWI Animal Health #86084-100-30) was administered with a subcutaneous injection at a dosage of 3.25 mg/kg once the mice were anesthetized.

The dorsal sides of the mice were shaved with an electric razor, and the area was cleaned with Povidone-iodine (7.5%) and 70% isopropyl alcohol. A 3-4 cm incision was made directly dorsal to the spine using a pair of sharp scissors. Next, a 1 cm incision was made in the muscles dorsal to the ovary. For animals receiving an ovariectomy, the ovary and ovarian horn were located, the ovarian horn was ligated, and the ovary was removed. Then, the same process was repeated contralaterally. For animals receiving a sham operation, both ovaries were located and replaced. The muscular layer was then closed using 4-0 absorbable internal sutures, and the skin was closed using external staples. External staples were removed 10-14 d after operations.

Immunostaining

Staining was performed according to standard procedures. Briefly, sections were air-dried for 30 min before removal of OCT via 3 rinses with PBS. Next, sections were treated with 0.5%-1% Triton100/PBS (Triton100, Sigma, T9284) solution depending on the position of the target gene. After 3 washes in 0.1% Tween20/PBS (PBST) (Tween20, Sigma, P7949), sections were incubated with a commercial blocking reagent (Abcam, ab126587) for 1 h followed by a primary antibody overnight at 4 °C. Sections were washed 3 times in PBST and then incubated with the Alexa-conjugated secondary antibody (Invitrogen, A-11008). Antibodies targeting the following proteins were used for immunostaining: Runx2 (1:100, Cell Signaling, D1L7F)Sp7 (1:100, Abcam, ab209484).

Histology

Femurs were harvested and fixed in 4% paraformaldehyde at room temperature overnight. Following decalcification in 10% EDTA for 5 wk, samples were passed through serial concentrations of ethanol for paraffin embedding. After sectioning at 12-16 μm using a microtome (Leica), hematoxylin and eosin staining was performed on deparaffinized sections following standard procedures.

For cryosections, decalcified samples were dehydrated gradually in 15% sucrose solution for 2-3 h, followed by 30% sucrose for 2-3 h, and 60% sucrose/OCT (Tissue-Tek, Sakura) (1:1) at 4 °C overnight. After being embedded in OCT compound, the samples were frozen in dry ice and sectioned at 12-14 μm thickness using a cryostat (CM1850; Leica).

MicroCT analysis

Femurs were harvested and fixed in 4% paraformaldehyde overnight. Samples were radiographed using a SCANCO μCT50 (Scanco V1.28) device at the University of Southern California Molecular Imaging Center. Images were collected at a resolution of 10-30 μm using a 70 kVp and 114 μA X-ray source. AVIZO 9.4.0 (Thermo Fisher Scientific) and VGStudio (Volume Graphics) were used to perform three-dimensional reconstruction. The voxel size used for all samples was 0.01 × 0.01 × 0.01 (10 μm). Measurements of bone volume fraction and trabecular number were performed at the region of the femur beginning at the distal epiphysis and extending 1 mm proximal. Sample sizes at 1 and 4 mo post tamoxifen were 4 and 3, respectively, for the sham-operated Runx2 control group, 5 and 6, respectively, for the ovariectomized Runx2 control group, 6 and 3, respectively, for the sham-operated Runx2 mutant mice, and 8 and 3, respectively, for the ovariectomized Runx2 mutant mice. Measurements of cortical width were performed 2 mm proximal to the epiphyseal plate. Sample sizes at 1 and 4 mo post tamoxifen were 5 and 5, respectively, for the sham-operated Runx2 control group, 8 and 6, respectively, for the ovariectomized Runx2 control group, 6 and 3, respectively for the sham-operated Runx2 mutant mice, and 10 and 6, respectviely, for the ovariectomized Runx2 mutant mice. Investigators were not blinded during measurements.

Von Kossa

Femurs were harvested and fixed in 4% paraformaldehyde at room temperature overnight. Samples were passed through serial concentrations of ethanol for paraffin embedding. After sectioning at 12-16 μm using a microtome (Leica), von Kossa staining was performed on deparaffinized sections following standard procedures. Briefly, samples were incubated in 1% Silver Nitrate (Abcam, 150 687) under a bright light (60 W) for 30 min. Next, samples were incubated in 5% sodium thiosulfate (Abcam, 150 687) for 5 min. Finally, samples were counterstained with Nuclear Fast Red (Abcam, 246 831) for 5 min and mounted.

RNAScope

Femurs were harvested and fixed in 4% paraformaldehyde at room temperature overnight. Following decalcification in 10% EDTA for 5 wk, samples were passed through serial concentrations of sucrose for embedding in OCT. After sectioning at 12-16 μm using a cryostat (Leica), RNAscope was performed according to the manufacturer’s protocol (Advanced Cell Diagnostics, 323100).

Sample size and statistics

SPSS version 27 and Microsoft Excel were used for statistical analysis. ANOVA and post-hoc comparisons was used to determine significance. A p-value < .05 was considered significant. The symbol “^*” indicates a p-value less than .05, and the symbol “^**” indicates a p-value less than .01.

Results

Runx2 expression in Gli1⁺ MSC-derived osteoprogenitors

To investigate the function of Runx2 in Gli1⁺ osteogenic lineage in adult bone tissue, we first examined the expression of Runx2, as well as colocalization of Runx2⁺ cells and Gli1⁺ progeny cells in adult mouse femurs. At 1 month (mo) after tamoxifen induction of 2-mo-old mice, Runx2 was expressed in prehypertrophic chondrocytes of the epiphyseal plate and the distal metaphysis. Its expression overlapped with Gli1⁺ cells in the femurs of adult mice (Figure 1A-D). These results indicate that Runx2 may play a critical role in regulating Gli1⁺ cells undergoing osteogenic differentiation.

Loss of Runx2 in Gli1⁺ cells mitigates cortical bone loss following ovariectomy

To further study whether Runx2 signaling is functionally required for adult bone homeostasis, we generated a Gli1Cre^ERT2;Runx2^fl/fl transgenic mouse model to specifically delete Runx2 from Gli1⁺ progeny (Figure S1). We found that the loss of Runx2 in Gli1⁺ progeny resulted in an increased width of the femoral cortical bone in adult mice, suggesting that Runx2 is indispensable for maintaining adult bone homeostasis (Figure 1E-H). More importantly, the increased width of the cortical bone observed in the femurs of adult Gli1Cre^ERT2;Runx2^fl/fl mice led us to hypothesize that the deletion of Runx2 may help protect against bone loss caused by postmenopausal osteoporosis. To test this hypothesis, we performed ovariectomies on both control and Runx2 mutant mice to attenuate estrogen production and model postmenopausal osteoporosis. The success of the ovariectomies was confirmed through uterine weight measurement (Figure S2). Operations were performed at 2 mo of age, and tamoxifen was administered 1 wk post-operatively to induce Runx2 deletion in Gli1Cre^ERT2;Runx2^fl/fl mice and as a control in Runx2^fl/fl mice. Samples were collected 1 and 4 mo post-tamoxifen induction. We analyzed the cortical bone thickness of sham-operated control mice (Runx2^fl/fl SHAM), ovariectomized control mice (Runx2^fl/fl OVX), sham-operated Gli1Cre^ERT2;Runx2^fl/fl mice (Gli1Cre^ERT2;Runx2^fl/fl SHAM), and ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice (Gli1Cre^ERT2;Runx2^fl/fl OVX) using microCT and H&E staining. Histological analysis showed that deletion of Runx2 resulted in a significant increase in cortical width in both Gli1Cre^ERT2;Runx2^fl/fl SHAM and Gli1Cre^ERT2;Runx2^fl/fl OVX groups at 1 mo post-tamoxifen induction compared to Runx2^fl/fl SHAM and Runx2^fl/fl OVX, respectively. Meanwhile, ovariectomy led to a slight but statistically insignificant decrease in femoral cortical width in the Runx2^fl/fl OVX group and Gli1Cre^ERT2;Runx2^fl/fl OVX group at 1 mo post-tamoxifen induction compared to Runx2^fl/fl SHAM and Gli1Cre^ERT2;Runx2^fl/fl SHAM, respectively (Figure 2A, C, E, G, and I). These results indicated that deletion of Runx2 led to increased cortical bone formation in the adult mouse femur. However, we recognized that 1 mo may not be sufficient for the development of osteoporosis induced by ovariectomy. Therefore, we collected samples at 4 mo post-tamoxifen induction for further comprehensive analysis. We found that Runx2^fl/fl OVX mice displayed a significant decrease in the width of the femoral cortical bone compared to Runx2^fl/fl SHAM mice (p=2.39x10^-4; Figure 2B, D, and I), and Gli1Cre^ERT2;Runx2^fl/fl OVX mice also showed a significant reduction in the width of the femoral cortical bone compared to Gli1Cre^ERT2;Runx2^fl/fl SHAM mice. However, within the ovariectomized groups, the reduction in femoral cortical bone width observed in Gli1Cre^ERT2;Runx2^fl/fl mice was not as significant as that observed in Runx2^fl/fl mice. Meanwhile, the cortical bone width of Gli1Cre^ERT2;Runx2^fl/fl OVX mice was significantly thicker than that of Runx2^fl/fl OVX mice (p=.039, Figure 2D, H, and I). These results suggest that although ovariectomy can cause bone loss in both Runx2^fl/fl and Gli1Cre^ERT2;Runx2^fl/fl mice, loss of Runx2 in Gli1⁺ cells can mitigate cortical bone loss following ovariectomy.

Loss of Runx2 in Gli1⁺ cells has no significant effect on bone strength

To further investigate whether the changes in the cortical bone we observed in these four groups affected bone strength, we conducted a 3-point bending test. We compared stiffness, maximum load, breaking displacement, and energy absorption across sham-operated and ovariectomized control and mutant mice (Runx2^fl/fl SHAM, Runx2^fl/fl OVX, Gli1Cre^ERT2;Runx2^fl/fl SHAM, and Gli1Cre^ERT2;Runx2^fl/fl OVX) (Figure 3). Operations were performed at 2 mo of age, and tamoxifen was administered 1 wk post-operatively. Samples were collected 1 mo post-tamoxifen induction for analysis. The 3-point bending tests showed that there was no significant difference in bone stiffness between sham-operated and ovariectomized controls, but there was a significant decrease in stiffness in Gli1Cre^ERT2;Runx2^fl/fl SHAM mice compared to ovariectomized control mice (p=.03, Figure 3A). This result indicated that the deletion of Runx2 from Gli1⁺ progeny can affect bone stiffness, potentially due to increased cortical bone thickness. There was also a significant increase in stiffness in Gli1Cre^ERT2;Runx2^fl/fl OVX mice compared to Gli1Cre^ERT2;Runx2^fl/fl SHAM mice, suggesting that ovariectomy, which causes a reduction in cortical bone width, could counteract the effect caused by the deletion of Runx2 (p=.011, Figure 3A). We did not observe significant differences across the four groups in maximum load, breaking displacement, or energy absorption (Figure 3). These results suggest that both Runx2 and ovariectomy play significant roles in affecting bone stiffness, but not other parameters of bone strength.

$Three-point bending test for femurs. (A) Bone stiffness, as measured by the resistance offered by the whole bone to the applied displacement in the elastic region. (B) Results of maximum load (also known as ultimate force), which is the maximum value of load attained during the test and indicates the bone strength. (C) Results of the displacement from the yield point to the fracture point, which is a measure of ductility. (D) Energy absorption represents the work that must be done to fracture the bone. Measurements are from four groups: SHAM-operated Runx2fl/fl mice (Runx2fl/fl SHAM), ovariectomized Runx2fl/fl mice (Runx2fl/fl OVX), SHAM-operated Gli1CreERT2;Runx2fl/fl mice (Gli1CreERT2;Runx2fl/fl SHAM), and ovariectomized Gli1CreERT2;Runx2fl/fl mice (Gli1CreERT2;Runx2fl/fl OVX). Samples were collected at 1 mo post-tamoxifen induction and n = 3 for each group. * indicates p<.05. Schematic illustration at the bottom indicates the time point of sample collection.$

Figure 3

Three-point bending test for femurs. (A) Bone stiffness, as measured by the resistance offered by the whole bone to the applied displacement in the elastic region. (B) Results of maximum load (also known as ultimate force), which is the maximum value of load attained during the test and indicates the bone strength. (C) Results of the displacement from the yield point to the fracture point, which is a measure of ductility. (D) Energy absorption represents the work that must be done to fracture the bone. Measurements are from four groups: SHAM-operated Runx2^fl/fl mice (Runx2^fl/fl SHAM), ovariectomized Runx2^fl/fl mice (Runx2^fl/fl OVX), SHAM-operated Gli1Cre^ERT2;Runx2^fl/fl mice (Gli1Cre^ERT2;Runx2^fl/fl SHAM), and ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice (Gli1Cre^ERT2;Runx2^fl/fl OVX). Samples were collected at 1 mo post-tamoxifen induction and n = 3 for each group. ^* indicates p<.05. Schematic illustration at the bottom indicates the time point of sample collection.

Loss of Runx2 in Gli1⁺ cells mitigates trabecular bone loss following ovariectomy

Trabecular bone loss is considered an early sign of osteoporosis.¹ To evaluate the effects of Runx2 deletion in Gli1⁺ progenitors on trabecular bone tissue homeostasis, we compared the trabecular morphology of sham-operated control mice (Runx2^fl/fl SHAM), ovariectomized control mice (Runx2^fl/fl OVX), sham-operated Gli1Cre^ERT2;Runx2^fl/fl mice (Gli1Cre^ERT2;Runx2^fl/fl SHAM), and ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice (Gli1Cre^ERT2;Runx2^fl/fl OVX) using microCT imaging. Operations were performed at 2 mo of age, and tamoxifen was administered 1 wk post-operatively. Samples were collected 1 and 4 mo post-tamoxifen induction for analysis. MicroCT imaging showed that trabecular bone mass was relatively unchanged across all groups at 1 mo post-tamoxifen induction (Figure 4A, C, E, G). Quantification analysis of trabecular bone volume fraction, trabecular thickness, trabecular number, and trabecular separation across the 4 groups further confirmed no significant differences (Figure 4I-L). At 4 mo post-tamoxifen induction, the ovariectomized control group showed a significant reduction in trabecular bone compared to the sham-operated control group (Figure 4B, D, and I). Detailed multi-parameter analysis showed significant reductions in trabecular bone volume fraction (p=2.06x10^-4) and trabecular number (p=.014) in the Runx2^fl/fl OVX group (Figure 4I and K). We also observed a significant increase in trabecular separation (p=3.48x10^-3) in the Runx2^fl/fl OVX group compared to the Runx2^fl/fl SHAM group (Figure 4L). These results showed that our ovariectomy model could cause bone mass loss and mimic the bone defects observed clinically in osteoporosis. When we compared the sham-operated mice, we found an increase in trabecular bone in the Gli1Cre^ERT2;Runx2^fl/fl SHAM group (Figure 4B, F, I, and J). Gli1Cre^ERT2;Runx2^fl/fl SHAM mice also showed a significant increase in trabecular bone volume fraction (p=5.38x10^-4) (Figure 4I) and trabecular thickness (p=2.84x10^-3) (Figure 4J) compared to sham-operated controls, indicating that loss of Runx2 causes increased trabecular bone formation during adult bone homeostasis. More importantly, ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice showed a significant increase in trabecular bone volume fraction (p=1.15x10^-7), trabecular thickness (p=1.41x10^-4) and trabecular number (p=1.85x10^-3), as well as a significant decrease in trabecular separation (p=2.72x10^-4) compared to the ovariectomized Runx2^fl/fl group (Figure 4D, H, and I-L). These results indicate that deletion of Runx2 can protect against trabecular bone volume loss following ovariectomy.

Figure 4

MicroCT analysis of femur trabecular bone. (A-H) MicroCT analysis of trabecular bone 1 and 4 mo after tamoxifen induction. Boxes in A-H are magnified in A′-H′, respectively. Scale bars: 1 mm. (I-L) Quantification of trabecular bone 1 mm proximal to the epiphyseal plate at 1mpt and 4mpt. BV/TV: Bone volume over total volume; Tb.Th: Trabecular thickness; Tb.N: Trabecular number; Tb.Sp: Trabecular separation. Data are mean ± SD. ^* indicates p<.05; ^** indicates p<.01. Schematic illustration at the bottom indicates the time point of sample collection.

To further evaluate changes in osteogenesis, we used von Kossa staining to examine trabecular bone mineralization in sham-operated Runx2^fl/fl mice, ovariectomized Runx2^fl/fl mice, sham-operated Gli1Cre^ERT2;Runx2^fl/fl mice and ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice 1 mo after surgery. The mineralized trabecular surface area decreased in ovariectomized Runx2^fl/fl mice compared to sham-operated Runx2^fl/fl mice (Figure 5A-D), indicating ovariectomy led to a decrease in trabecular bone mineralization. We then analyzed trabecular bone mineralization in sham-operated and ovariectomized Gli1Cre^ERT2;Runx2^fl/fl animals. Compared to the control OVX group (Figure 5C and D), we found increased trabecular mineralization in both Gli1Cre^ERT2;Runx2^fl/fl groups (Figure 5E-H), suggesting deletion of Runx2 from Glil1⁺ cells can protect adult mice from osteoporosis-induced trabecular bone loss. These results further confirmed that loss of Runx2 leads to an increase in trabecular bone mineralization and protects against trabecular bone loss following ovariectomy.

Figure 5

Loss of Runx2 increases femoral mineralization. Von Kossa staining of (A-B) sham-operated Runx2^fl/fl mice, (C-D) ovariectomized Runx2^fl/fl mice, (E-F) sham-operated Gli1Cre^ERT2;Runx2^fl/fl mice, and (G-H) ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice 1 mo after tamoxifen induction. Boxes in A, C, E, and G are magnified in B, D, F, and H, respectively. Schematic illustration at the bottom indicates the time point of sample collection. Scale bars: 1 mm.

Loss of Runx2 in Gli1⁺ cells affects epiphyseal morphology

To investigate whether deletion of Runx2 affects bone tissue homeostasis through interruption of the epiphyseal plate, we examined epiphyseal morphology in our samples carefully. The epiphyseal plate region was compared across Runx2^fl/fl SHAM, Runx2^fl/fl OVX, Gli1Cre^ERT2;Runx2^fl/fl SHAM, and Gli1Cre^ERT2;Runx2^fl/fl OVX groups 1 mo post-tamoxifen induction. We observed no significant changes in the overall width of the epiphyseal plate following Runx2 deletion or ovariectomy (Figure 6A, C, E, and G). However, the morphology and arrangement of chondrocytes within the epiphyseal plate showed notable differences in ovariectomized and Runx2 mutant mice (Figure 6B, D, F, and H). We found that the chondrocyte nuclei were smaller in the Runx2^fl/fl OVX group, accompanied by an abnormal arrangement of cells (Figure 6D). This result indicates that ovariectomy can affect the epiphyseal plate of the femur in adult mice. Mild defects in the epiphyseal plate were also observed in Gli1Cre^ERT2;Runx2^fl/fl SHAM (Figure 6F). The epiphyseal plate showed the most severe defects in the Gli1Cre^ERT2;Runx2^fl/fl OVX group (Figure 6H). To summarize, both the loss of Runx2 in Gli1⁺ progenitors and ovariectomy impact epiphyseal homeostasis.

Figure 6

Loss of Runx2 affects femur epiphyseal morphology. (A-H) H&E staining of 1 mpt samples. Boxes in A, C, E, and G are magnified in B, D, F, and H, respectively. Dashed lines in A, C, E, and G indicate epiphyseal plate. Arrows in B, D, F, and H indicate representative chondrocytes in the epiphyseal zone. Scale bars: 100 μm. Schematic illustration at the bottom indicates the time point of sample collection.

Loss of Runx2 in Gli1⁺ cells results in decreased osteoclast activity

Bone homeostasis is a dynamic equilibrium involving the participation of osteoblasts and osteoclasts. Therefore, we first investigated whether loss of Runx2 in Gli1⁺ progeny affected bone tissue homeostasis through osteoclast activity. By analyzing the expression of the osteoclast marker Ctsk (Cathepsin-K), we compared osteoclast activity across Runx2^fl/fl SHAM, Runx2^fl/fl OVX, Gli1Cre^ERT2;Runx2^fl/fl SHAM, and Gli1Cre^ERT2;Runx2^fl/fl OVX mice. Operations were performed at 2 mo of age, and tamoxifen was administered 1 wk post-operatively. Samples were collected 1 and 4 mo post-tamoxifen induction. At 1 mo post-tamoxifen induction, we observed a significant decrease in Ctsk expression in the Gli1Cre^ERT2;Runx2^fl/fl SHAM group compared to the Runx2^fl/fl SHAM group (Figure 7A, C, E, G, and Q). The expression of Ctsk in Gli1Cre^ERT2;Runx2^fl/fl OVX mice was also slightly less than in Runx2^fl/fl OVX controls at 1 mo post-tamoxifen induction (Figure 7B, D, F, H, and Q). These data suggested that deletion of Runx2 from Gli1⁺ cells reduced osteoclast activity. At 4 mo post-tamoxifen induction, we observed a significant increase in Ctsk expression in ovariectomized groups compared to the sham-operated groups (Figure 7I-P). This suggested that ovariectomy can induce osteoclast activity, which might exacerbate bone loss. In addition, we observed decreased expression of Ctsk in the Gli1Cre^ERT2;Runx2^fl/fl SHAM group compared to the Runx2^fl/fl SHAM group (Figure 7I, K, M, O, and Q). More importantly, a significant decrease in Ctsk expression was observed in the Gli1Cre^ERT2;Runx2^fl/fl OVX group compared to the Runx2^fl/fl OVX group at 4 mo post-tamoxifen induction (Figure 7J, L, N, P, and Q). These data suggested that loss of Runx2 in Gli1⁺ cells can protect against ovariectomy-induced bone loss by reducing osteoclast activity.

Figure 7

Deletion of Runx2 results in decreased expression of osteoclast marker Ctsk. (A-H) Expression of Ctsk in 1mpt mouse femurs. Boxes in A-D are magnified in E-H, respectively. Arrowheads in E-H indicate representative Ctsk⁺ cells. Dashed lines in E-H indicate epiphyseal plate. (I-P) Expression of Ctsk in 4mpt mouse femurs. Boxes in I-L are magnified in M-P, respectively. Arrowheads in M-P indicate representative Ctsk⁺ cells. Dashed lines in M-P indicate epiphyseal plate. (Q) The percentage of Ctsk + area across samples is analyzed using ANOVA. ^* indicates p<.05. Scale bars: 200 μm. Schematic illustration at the bottom indicates the time point of sample collection.

Loss of Runx2 in Gli1⁺ cells leads to a decrease in osteoblast expression

To further investigate whether Runx2 plays a critical role in regulating osteoblasts during adult bone tissue homeostasis, we compared the expression of Sp7, a marker for osteoblasts, across Runx2^fl/fl SHAM, Runx2^fl/fl OVX, Gli1Cre^ERT2;Runx2^fl/fl SHAM, and Gli1Cre^ERT2;Runx2^fl/fl OVX mice. Operations were performed at 2 mo of age, and tamoxifen was administered 1 wk post-operatively. Samples were collected 1 and 4 mo post-tamoxifen induction. We found no significant change in the expression of Sp7 between sham-operated and ovariectomized control mice or between sham-operated and ovariectomized mutant mice at 1 mo after tamoxifen induction (Figure 8A-D, I). However, decreased expression of Sp7 was observed in Gli1Cre^ERT2;Runx2^fl/fl SHAM mice at the location below the epiphyseal plate compared to Runx2^fl/fl SHAM mice at 4 mo after tamoxifen induction (Figure 8E, G, and I). Similarly, Gli1Cre^ERT2;Runx2^fl/fl OVX mice exhibited less Sp7 than Runx2^fl/fl OVX controls at 4 mo after tamoxifen induction (Figure 8F, H, and I). Together, these data indicated that Runx2 plays a role in regulating osteoblasts during adult bone homeostasis and that bone loss observed in ovariectomized groups is not caused by abnormal osteoblast activity. Furthermore, when comparing sham-operated control and mutant mice, Ctsk staining revealed a significant decrease in osteoclast activity as early as 1 mo after tamoxifen induction, while osteoblast activity, indicated by Sp7, remained unchanged. This suggests that activity of osteoclasts may be more important than that of osteoblasts in the development of the thicker cortical bone observed in Gli1Cre^ERT2;Runx2^fl/fl mice.

Figure 8

Deletion of Runx2 results in a slight decrease in osteoblast expression at 4mpt. (A-D) Expression of Sp7 in sham-operated Runx2^fl/fl mice, ovariectomized Runx2^fl/fl mice, sham-operated Gli1Cre^ERT2;Runx2^fl/fl mice, and ovariectomized Gli1Cre^ERT2;Runx2^fl/fl mice 1 mo after tamoxifen induction. (E-H) Expression of Sp7 at 4 mo after tamoxifen induction across the groups. Dashed lines indicate epiphyseal plate, and arrowheads indicate representative positive signals. Scale bars: 200 μm.

10.5152/eurjrheum.2016.048

Discussion

Runx2 is a key regulator of osteogenesis during development. In this study, we first showed that ovariectomy leads to a decrease in cortical and trabecular bone mass in adult mice. Next, we demonstrated that loss of Runx2 in Gli1⁺ cells of adult mice leads to an increase in bone mass. This increase in bone mass mitigated bone loss associated with ovariectomy. We also found that the increase in bone observed following conditional deletion of Runx2 was caused by a reduction in osteoclasts. Together, these results indicate that inactivation of Runx2 increases bone formation and protects against bone loss in this the model of estrogen-deficient osteoporosis via downregulation of osteoclasts.

Our findings represent an interesting effect of Runx2 disruption on bone tissue homeostasis in adults. During embryonic development, disruption of Runx2 prevents ossification by inhibiting osteoblast differentiation and results in a complete inhibition of bone formation.⁶ In the relatively small number of studies that have manipulated Runx2 expression in postnatal and adult mice, the effects of Runx2 disruption on bone homeostasis have been unclear.⁹ Global deletion of Runx2 at 4 wk of age results in decreased bone mass and increased bone marrow adiposity.⁷ Similarly, decreased Runx2 expression caused by knockdown of the Runx2 promoter gene Gata4 has been correlated with reduced bone mineralization.¹⁰ Upregulation of Runx2, induced by L. helveticus supplementation, has been correlated with an increase in bone density in ovariectomized adult rats.¹¹ Here, we show that loss of Runx2 in Gli1⁺ cells protects bone tissue from turnover following ovariectomy in adult mice. However, it has also been shown that overexpression of Runx2 in osteoblastic lineage cells leads to osteopenia in aging mice due to elevated bone turnover and reduced bone mineralization.¹² Our findings in the context of these prior studies suggest that Runx2 deletion may have specific beneficial effects on bone formation when targeted to Gli1⁺ cells and their progeny.

Runx2 is known to regulate osteoblast production of RANKL and OPG, which are essential for regulating osteoclast expression.^13–15 RANKL promotes osteoclast differentiation in the presence of M-CSF, and OPG inhibits osteoclast differentiation. Runx2 deficient mice have decreased osteoclastogenesis in vivo,⁶ and Runx2-mutant macrophage precursors display decreased differentiation into osteoclasts in vitro.¹⁵ Runx2 deficiency in calvaria-derived osteoblasts has been reported to increase secretion of OPG and suppress secretion of RANKL, resulting in an inhibition of osteoclast differentiation.¹⁶ It has also been shown that osteoblastic cells of adult mice overexpressing Runx2 demonstrate increased expression of RANKL and collagenase 3,¹² as well as an increase in the ratio of RANKL to OPG,¹⁷ both of which promote elevated osteoclast formation. Together, these studies suggest that Runx2 promotes osteoclastogenesis. Our findings of increased bone mass and decreased osteoclast expression following deletion of Runx2 support the role of Runx2 as a promoter of osteoclastogenesis. However, Runx2 has been reported to activate transcription of an OPG promoter in osteoblast cell lines.¹⁸ Further studies are required to understand the role of Runx2 in osteoblast signaling and RANKL and OPG expression in adult mice.

Runx2 is also known to promote maturation of osteoblasts.⁶ It has been shown that it is essential for the proliferation of osteoblast precursors in the calvaria, promoting proliferation primarily through upregulation of Fgfr2.¹⁹ Additionally, Runx2 signaling regulates differentiation of multipotent mesenchymal stem cells into mature osteoblasts through reciprocal regulation with Fgf, hedgehog, Wnt, Pth1h, and Sp7 signaling pathways.²⁰ Our finding that the loss of Runx2 results in a slight decrease in osteoblast marker expression supports its role as a promoter of osteoblast expression.

One of the main strengths of our study is the use of a mouse model with inducible, targeted, temporally-controlled deletion of Runx2. This allowed us to precisely time the inactivation of the gene in relation to ovariectomy and restrict deletion of Runx2 to osteoprogenitors and their progeny. This model allowed us to examine the role of Runx2 in adult bone tissue homeostasis. Future studies will elucidate the mechanisms underlying the protective effects of Runx2 deletion, including the role of Runx2 in osteoclast expression and RANKL and OPG regulation in adult bone tissue. One weakness of this study was the timing of the sham and ovariectomy procedures. Mice do not achieve skeletal maturity until they reach 12-16 wk of age. All operations were performed when mice were 2 mo old, prior to the time at which they would be expected to reach skeletal maturity. The timing of these procedures may have affected the skeletal development of the mice and disrupted our measurements of bone homeostasis.

In summary, our study has revealed that Runx2 deletion protects adult mice from reductions in bone mass and mineralization following ovariectomy. This effect is directionally opposite that observed with Runx2 deficiency in developing embryos, suggesting that Runx2 may have a special role in regulating bone tissue homeostasis in adult mice.

Acknowledgments

We acknowledge Dr. Bridget Samuels for critical editing of the manuscript and the USC Molecular Imaging Center (MIC) for conducting microCT scans of tissue samples. We also thank Sally Anderson for her technical support. This study was supported in part by funding from the University of Southern California.

Author contributions

Connor Buchanan (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing—original draft, Writing—review & editing), Shuo Chen (Conceptualization, Data curation, Investigation, Methodology), Yuan Yuan Investigation), Tingwei Guo (Conceptualization, Data curation, Formal analysis, Investigation, Project administration, Validation, Writing—review & editing), Jifan Feng (Data curation, Investigation, Project administration, Writing—review & editing), Mingyi Zhang (Formal analysis, Investigation, Project administration, Supervision, Writing—review & editing), Grace CareyInvestigation (Ishmael HowardInvestigation), Janet Sanchez (Investigation), Thach-Vu Ho (Methodology, Project administration), and Yang Chai (Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing—review & editing)

Funding

This study was supported in part by funding from the University of Southern California.

Conflicts of interest

The authors declare that there is no conflict of interest.

Data availability

We will make all data available upon request.

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