Conceived and designed the experiments: KM RD. Performed the experiments: KM SIM. Analyzed the data: KM. Contributed reagents/materials/analysis tools: KM. Wrote the paper: KM. Wrote the manuscript with critical input from all authors: TA. Planned and performed most of the experiments with assistance from CRM, HD, MN, XHP: KSM. Planned and performed nanoindentation analyses: CC. Performed XTM: GB. Participated in study design: ES. Administered drugs and did DXA measurements: HD RM. Performed CFU-F & CFU-OB: MN. Did the histology: XHP. Planned and performed gene expression studies: DN TA. Performed microCT: WH JB. Participated in study design: DW. Planned fracture toughness tests and X-ray tomography, which were performed by SSI-M and GB respectively: RR. Planned micro-tomography and macromechanical tests, which were performed by JWB and WRH: LS. Designed and coordinated the study and supervised all experiments: TAG TA.
The authors have declared that no competing interests exist.
During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-β has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-β signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-β signaling on bone remain unclear. To examine the role of TGF-β in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-β type I receptor (TβRI) kinase on bone mass, architecture and material properties. Inhibition of TβRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TβRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TβRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TβRI inhibitors may be effective in treating conditions of skeletal fragility.
In skeletal development, each bone is formed with a distinctive size, geometry, architecture, and material properties. Among the many growth factors and hormones involved in this process
The effects of postnatal manipulation of TGF-β signaling on bone mass and quality are difficult to predict based on developmental studies. For example, osteoporosis and bone fragility are observed in mice with increased TGF-β production
The recent development of specific inhibitors of the TGF-β type I receptor (TβRI) kinase that block most if not all TGF-β signaling events
Maintenance of the postnatal skeleton depends on the functional coordination between bone-depositing osteoblasts and bone-resorbing osteoclasts
In the current study, we found that the TβRI kinase inhibitor, SD-208, affects osteoblast and osteoclast function to coordinately regulate several bone parameters, resulting in increased bone mass and trabecular bone volume, as well as increased mineral concentration and elastic modulus of bone matrix. This was associated with an increased resistance to vertebral fracture. These results suggest that pharmacologic inhibition of TGF-β signaling may have therapeutic utility in a variety of bone diseases characterized by poor bone quality, low bone mass and a propensity to fracture.
To determine the effects of pharmacologic inhibition of TGF-β signaling on bone, mice were treated for 6 weeks with either of two doses of SD-208, a small molecule that blocks ATP binding to the type I TGF-β receptor to specifically inhibit its kinase activity
Five hours after TGF-β administration, SBE-Luc mice showed increased bioluminescence on the dorsal and ventral surfaces of the head where relatively little superficial tissue covers skeletal elements (calvarial bone and jaws) (a). Mice pretreated with SD-208 showed less basal and TGF-β-inducible luminescence than vehicle-treated controls (a, lower panels). SD-208 also inhibited reporter activity in SBE-Luc mouse calvarial explants cultured overnight with TGF-β (b). SD-208 treatment of calvarial explants inhibits expression of the TGF-β-inducible gene, PAI-1
Longitudinal examination of the bone mineral density (BMD) by dual energy X-ray absorptiometry (DXA) showed the normal increase in BMD between 1 and 2.5 months of age. Accordingly, vehicle-treated male and female mice showed an increase of 21.8% and 29.6%, respectively, in whole body BMD after 6 weeks (
DXA was used to measure BMD longitudinally for male (a, c, e, g) and female mice (b, d, f, h) treated with or without the TβRI inhibitor SD-208 at 20 mg/kg or 60 mg/kg. SD-208 treatment at the 60 mg/kg dose caused an increase in total body (a, b) tibia (c, d), femur (e, f), and lumbar spine (g, h) BMD. SD-208 at the 20 mg/kg dose increased femoral BMD in female mice (f). Data represent mean±SEM (p<0.05, as determined by two-way analysis of variance (ANOVA).
More pronounced effects were apparent in the tibia and femur, where the BMD was already significantly increased within 3 weeks of SD-208 treatment relative to vehicle-treated controls (
To determine if the increased BMD resulted from changes in cortical or trabecular bone, dissected femora and tibiae were analyzed using micro-computed tomography (micro-CT). Reconstructed images of trabecular bone in the distal femur showed a dose-dependent increase in trabecular bone volume following 6 weeks of SD-208 treatment in both male and female mice (
Micro-CT images show increased femoral trabecular bone volume following SD-208 treatment in male and female mice, relative to vehicle-treated controls (a). Quantitative analyses show that SD-208 increased trabecular bone volume (BV/TV, fraction) (b), connectivity density (c), and trabecular number (d), but decreased trabecular spacing (e) in male and female femora. Data represent mean±SEM (p<0.05, as determined by one-way ANOVA Newman-Keuls multiple comparison test).
Male | Female | |||||||||||
Tibia | Femur | Tibia | Femur | |||||||||
Vehicle | 20 mg SD-208 | 60 mg SD-208 | Vehicle | 20 mg SD-208 | 60 mg SD-208 | Vehicle | 20 mg SD-208 | 60 mg SD-208 | Vehicle | 20 mg SD-208 | 60 mg SD-208 | |
0.11±0.006 | 0.182±0.018** | 0.20±0.009** | 0.132±0.017 | 0.169±0.013* | 0.208±0.008** | 0.09±0.009 | 0.113±0.007 | 0.161±0.014** | 0.048±0.009 | 0.071±0.0009 | 0.175±0.011*** | |
0.048±0.0006 | 0.049±0.002 | 0.047±0.0004 | 0.049±0.0012 | 0.049±0.003 | 0.046±0.0003 | 0.045±0.002 | 0.048±0.0009 | 0.046±0.0005 | 0.039±0.002 | 0.045±0.001* | 0.048±0.0005* | |
4.44±0.19 | 5.53±0.10*** | 6.19±0.15*** | 4.82±0.29 | 5.32±0.09 | 6.27±0.18** | 3.34±0.15 | 3.69±0.15 | 5.22±0.39** | 3.54±0.08 | 3.99±0.17 | 5.43±0.16*** | |
0.20±0.01 | 0.16±0.004 | 0.15±0.003* | 0.20±0.01 | 0.18±0.002 | 0.14±0.004** | 0.30±0.014 | 0.26±0.012 | 0.11±0.016*** | 0.28±0.006 | 0.25±0.011 | 0.18±0.018** | |
60.52±4.13 | 129.6±12.06** | 197.3±15.16*** | 90.32±1.61 | 163.1±8.68*** | 264.3±16.09*** | 47.13±5.21 | 50.55±6.01 | 138.2±25.01** | 25.7±6.13 | 33.48±4.95 | 175.2±17.97*** | |
2.36±0.06 | 1.98±0.13 | 1.97±0.082 | 2.39±0.11 | 2.07±0.04* | 1.78±0.009*** | 2.33±0.07 | 2.15±0.12 | 2.34±0.044 | 3.28±0.14 | 3.21±0.048 | 2.09±0.106*** | |
2.13±0.06 | 2.11±0.07 | 1.92±0.06 | 1.33±0.009 | 1.42±0.04 | 1.33±0.03 | 2.34±0.066 | 2.6±0.023** | 2.01±0.089*** | 1.4±0.106 | 1.4±0.006 | 1.42±0.006 |
Micro-computed tomography was used to assess several quantitative parameters of trabecular bone structure. The mean values and standard deviations are presented here. Significant differences between vehicle and SD-208 treated groups are indicated (*p<0.05, **p<0.01, ***p<0.001).
Male | Female | |||||
Vehicle | SD-208 20 mg | SD-208 60 mg | Vehicle | SD-208 20 mg | SD-208 60 mg | |
0.183±0.019 | 0.182±0.016 | 0.172±0.007 | 0.155±0.007 | 0.156±0.006 | 0.152±0.003 | |
0.201±0.005 | 0.178±0.006 | 0.186±0.005 | 0.186±0.011 | 0.195±0.005 | 0.188±0.004 | |
0.329±0.038 | 0.346±0.035 | 0.405±0.025 | 0.281±0.017 | 0.285±0.015 | 0.281±0.007 | |
1.057±0.098 | 1.202±0.081 | 0.986±0.057 | 0.964±0.047 | 0.977±0.045 | 0.958±0.017 | |
0.592±0.017 | 0.546±0.017 | 0.577±0.004 | 0.554±0.012 | 0.555±0.005 | 0.560±0.010 | |
0.182±0.040 | 0.218±0.098 | 0.214±0.019 | 0.105±0.009 | 0.109±0.008 | 0.110±0.005 | |
0.761±0.078 | 0.891±0.233 | 0.729±0.055 | 0.571±0.049 | 0.596±0.038 | 0.614±0.020 | |
0.447±0.054 | 0.427±0.077 | 0.551±0.013 | 0.352±0.008 | 0.351±0.003 | 0.343±0.011 |
Micro-computed tomography was used to assess several quantitative parameters of cortical bone structure. The mean values and standard deviations are presented here.
Increased BMD may be due to increased osteoblast activity, reduced osteoclast activity or both. Quantitative histomorphometry confirmed the SD-208 dose-dependent increase in trabecular bone that was observed by micro-CT (
Representative H&E stained sections of femoral bone show the SD-208-dependent increase in trabecular bone in male and female mice (a). Histomorphometry shows that SD-208 increases trabecular bone volume in the femur (b) and tibia (data not shown), as well as osteoblast number (c) in a dose-dependent manner for male and female mice. Osteoclast numbers are reduced by SD-208 (60 mg/kg) in male mice (d). Dynamic histomorphometry of male mouse lumbar vertebrae shows that SD-208 treatment (60 mg/kg) increased mineral apposition rate (MAR) (e) and bone formation rate (BFR) (f). Data represent mean±SEM (*p<0.05, **p<0.01, ***p<0.001, as determined by one-way ANOVA Newman-Keuls multiple comparison test).
These data suggest that inhibition of TGF-β signaling increases bone mass by enhancing bone formation and inhibiting bone resorption. Dynamic histomorphometry revealed that SD-208 stimulates a dose-dependent increase in the mineral apposition rate and bone formation rate in male mice (
To determine if the changes in osteoblast and osteoclast numbers and activity resulted from changes in cell differentiation, bone marrow stromal cells that were isolated from vehicle- and SD-208-treated mice were examined ex vivo in osteoblast or osteoclast differentiation assays (
Bone marrow isolated from male and female mice treated with SD-208 (60 mg/kg) has increased numbers of osteoblast colony forming units (CFU-OB) (a) with no change in the number of colony forming units (CFU-F) (b). The number of TRAP-positive multinucleated cells (TRAP+ MNC) is lower in cultures from SD-208 treated mice than from vehicle-treated controls (c). Primary calvarial osteoblasts treated with TβRI-inhibitor SB431542 (10µM) or vehicle for 48 h show altered mRNA expression of PAI-1 (d) and several osteoblast and osteoclast regulatory factors including Runx2 (d), RANKL and OPG (e), and ephrinB2 and EphB4 (f). Data represent mean±SEM (*p<0.05, **p<0.01, ***p<0.001, as determined by unpaired
To investigate the effect of TβRI inhibitors on the expression of osteoblast and osteoclast regulatory factors, we utilized primary calvarial osteoblasts, which retain the capacity to differentiate into mineralizing osteoblasts, and have an intact autocrine TGF-β regulatory pathway. As in calvarial explants treated with SD-208 (
RANK ligand (RANKL) promotes osteoclast differentiation, function and survival
Recently, ephrin B2 and EphB4, a transmembrane ligand and receptor respectively, have been implicated as factors that couple osteoblast and osteoclast activities in bone metabolism
The net effect of TβRI inhibitors on bone is increased BMD, which reflects both bone mass and mineral concentration (
Analysis of each pixel from XTM scans of femora show that SD-208 (60 mg/kg) increases bone matrix mineral concentration with a mean of 1.90 g/cm3+/−0.066, relative to a mean mineral concentration of 1.54 g/cm3+/−0.069 for vehicle-treated controls (p<0.05, as determined by unpaired
Mineral concentration is a major determinant of bone matrix material properties
Treatment with the TβRI inhibitor SD-208 affects bone on several levels, including bone mass (
Male Vertebrae | ||
Vehicle | SD-208 60 mg | |
41.55±3.34 | 54.95±5.45* | |
98.61±11.52 | 111.1±18.70 |
Mean values±SEM for macromechanical tests of vertebral peak load and stiffness are shown. The significance of differences between vehicle and SD-208 (60 mg/kg) treated groups is indicated with p values (*p<0.05).
Male Femora | ||
Vehicle | SD-208 60 mg | |
16.09±0.90 | 15.55±0.52 | |
40.32±5.59 | 47.10±1.40 | |
4.367±0.783 | 4.755±0.509 |
Mean values±SEM for macromechanical tests of femoral peak load, stiffness and fracture toughness are shown.
Here we explored the role of TGF-β signaling in postnatal bone by systemic administration of a TβRI inhibitor to mature mice. Pharmacologic inhibition of TGF-β signaling resulted in dose-dependent increases in BMD, trabecular microarchitecture, bone matrix elastic modulus and mineral concentration. These coordinated changes in bone mass and parameters of bone quality improved the ability of vertebral bone to resist fracture. By targeting key regulatory pathways in osteoblasts and osteoclasts, TβRI inhibitors increased the number of osteoblasts and the bone formation rate, while reducing osteoclast numbers. Therefore, TβRI inhibition elicits both anabolic and anti-catabolic activities to improve bone quality.
The TβRI inhibitor-dependent increase in tibial BMD exceeded the physiologic increase in BMD over this time period or those induced by comparable regimens utilizing clinically available bisphosphonates or PTH
Some effects of TβRI inhibition on bone resulted from the reduction in osteoclast numbers and differentiation potential in SD-208-treated mice (
Treatment of mice with TβRI inhibitors resulted in increased osteoblast numbers and differentiation, and increased bone formation. Consistent with these data, reduced TGF-β signaling in Smad3+/− mice or DNTβRII mice also relieves the suppression of osteoblast differentiation by TGF-β, which is exerted by Smad3 and histone deacetylases
Ultimately, the ability of bone to resist fracture is the most clinically desirable outcome
In conclusion, pharmacologic inhibition of TGF-β signaling in postnatal bone increases bone quality. Coupling of osteoblast and osteoclast activity may be critical for the ability of TGF-β to coordinately control bone mass, architecture, and the material properties of bone. Therefore, therapies that produce a reliable reduction in TGF-β signaling may have significant clinical benefit in the treatment of diseases characterized by low bone mass and bone fragility. However, TβRI-inhibition may be counter-indicated for the treatment of existing bone fractures, where TGF-β plays a role in fracture repair. Additional studies evaluating the efficacy and potential sex-specificity of the mature skeletal response to TβRI inhibitors, particularly in ovariectomized animals, would be needed to determine their potential therapeutic value for post-menopausal osteoporosis. Careful consideration of safety is essential, given the critical role of TGF-β in normal physiological processes including the control of cell proliferation, differentiation, and apoptosis in many tissues.
In all studies, mice were handled and euthanized in accordance with approved institutional, national and international guidelines.
Four-week old male and female C57BL/6 mice were treated for 6 weeks with vehicle (1% methylcellulose) or SD-208 (20 mg/kg once daily or 60 mg/kg twice daily) by gavage. As described, SD-208 is a specific inhibitor of the TGF-β type I receptor, developed by Scios, Inc.
BMD was measured using a PIXImus mouse densitometer (GE Lunar II, Faxitron Corp., Wheeling, IL) (N = 15/group). Total body measurement was performed excluding the calvarium, mandible and teeth. Regions of interest were defined as the distal femur and proximal tibia just beneath the growth plate (12×12 pixels) and the lower lumbar spine (20×50 pixels). Values were expressed as percentage change in BMD over the pretreatment scan.
For demineralized bone histomorphometry, tissues were fixed for 48 h in 10% formalin, demineralized in 10% EDTA for 2 weeks, and embedded in paraffin to generate 3.5 µm longitudinal sections. Trabecular bone volume of the secondary spongiosa (BV/TV%) and osteoblast number (N.Ob/high power field) were measured on hematoxylin and eosin stained sections of the distal femur, proximal tibia, and lumbar vertebrae (N≥12 mice/group). Tartrate resistant acid phosphatase (TRAP) stained sections were used to quantify osteoclast number (N.Oc/BS/mm). Dynamic bone histomorphometry was performed on 7 µm thick sections of mineralized lumbar vertebrae embedded in methylmethacrylate using standard procedures. The mineral apposition rate (MAR, µm/day) and bone formation rate (BFR/BS, µm3/µm2/day) were measured on vertebral trabecular bone using fluorescence microscopy to visualize calcein labels as described
Formalin fixed tibiae and femora were imaged with micro-CT using a microCT-40 (Scanco Medical AG, Bassersdorf, Switzerland) using a voxel size of 12 µm in all dimensions (N≥12 mice/group). The region of interest comprised 240 transverse CT slices representing the entire medullary volume with a border lying approximately 100 µm from the cortex
The CT images of the mid-diaphysis of the tibia were segmented into bone and marrow regions by applying a visually chosen, fixed threshold for all samples, after smoothing the image with a three-dimensional Gaussian low-pass filter. The outer contour of the bone was found automatically with the built-in Scanco iterative contouring tool. Total area (TA) was calculated by counting all voxels within the contoured bone area, (BA) by counting all voxels that were segmented as bone, and marrow area (MA) was calculated as TA-BA. This calculation was performed on all 30 slices (1 slice = 12.5 µm), using the average for the final calculation. The outer and inner perimeter of the cortical midshaft was determined by a three-dimensional triangulation of the bone surface (BS) of the 30 slices, and cortical parameters were calculated as described
Bone marrow stromal cells were flushed from 6 femora and tibiae per treatment group, collected by centrifugation (1500 rpm, 10 minutes), resuspended (αMEM, 10% FCS), and incubated for 2 h at 37°C. For osteoblast assays, cells were cultured in αMEM, 15% FBS, 50 µg/ml ascorbic acid, and 10 mM β-glycerophosphate. The number of alkaline phosphatase-positive osteoblast progenitor forming colonies (CFU-F) and Alizarin Red-positive osteoblast forming colonies (CFU-OB) was quantified microscopically after 9 or 28 days of culture, respectively, as described
Calvarial explants were isolated from 10 day old SBE-Luc mice and cultured overnight in DMEM supplemented with 10% fetal bovine serum and 5 ng/ml TGF-β1 in the presence of either 150 nM SD-208 or an equivalent volume of vehicle (1% methylcellulose). Following culture, explants were moved to media containing luciferin (150 mg/ml) for immediate visualization of luciferase reporter activity with a bioluminescent imaging system (Xenogen). Explants were then crushed in liquid nitrogen using a mortar and pestle prior to additional tissue disruption in Trizol with a Omni-GLH homogenizer (Omni Scientific). Following Trizol extraction, RNA was further purified using RNeasy columns (Qiagen).
Primary calvarial osteoblasts were isolated from 3 to 5-day old mice and cultured in osteogenic conditions as described
XTM studies were used to assess the degree of mineralization of the bone; procedures were based on the work of Kinney
Dissected male mouse tibiae were embedded in a two-component epoxy resin (Stycast 1266) prior to sectioning with a precision low-speed saw to generate mid-tibial cortical bone surfaces for nanoindentation. A nanoindenter (Triboindenter, Hysitron, Minneapolis, MN) with a Berkovich tip was used to evaluate polished samples (0.25 µm) under dry conditions as described
Whole bone strength and load to failure were determined by mechanical testing of vertebrae and intact tibiae for at least 12 mice per treatment group as previously described
Fracture toughness testing was performed on at least 10 isolated femora per condition. Thawed samples were notched using a razor blade followed by a micronotching technique. Notches were evaluated to ensure that they were through-wall but notched less than 1/3 of the bone diameter. Samples were tested in 37°C HBSS in a three-point bending configuration with a custom-made rig for the ELF 3200 mechanical testing machine (ELF3200, Bose, EnduraTEC, Minnetonka, MN), in general accordance with ASTM Standard E-399 and E-1820
The diaphysis is not filled by trabecular bone following SD-208 treatment. Although increased trabecular bone in femora from SD-208-treated mice (60 mg/kg) is evident in reconstructed micro-CT images, the trabecualr bone does not extend past the distal third of the femur. The scale bar is 1 mm.The diaphysis is not filled by trabecular bone following SD-208 treatment. Although increased trabecular bone in femora from SD-208-treated mice (60 mg/kg) is evident in reconstructed micro-CT images, the trabecualr bone does not extend past the distal third of the femur. The scale bar is 1 mm.
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Thank you to E. Chin, S. Provot, and M. Nakamura for their contributions. XTM was performed at the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the Office of Science, U.S. Department of Energy (DE-AC02-05CH11231).