1. Cell Biology

Cortical bone maturation in mice requires SOCS3 suppression of gp130/STAT3 signalling in osteocytes

  1. Emma C Walker
  2. Kim Truong
  3. Narelle E McGregor
  4. Ingrid J Poulton
  5. Tsuyoshi Isojima
  6. Jonathan H Gooi
  7. T John Martin
  8. Natalie A Sims  Is a corresponding author
  1. St. Vincent’s Institute of Medical Research, Australia
  2. University of Melbourne, Department of Medicine at St. Vincent’s Hospital, Australia
  3. Department of Pediatrics, Teikyo University School of Medicine, Japan
  4. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Australia
https://doi.org/10.7554/eLife.56666
Share this article
Cite this article
  1. Emma C Walker
  2. Kim Truong
  3. Narelle E McGregor
  4. Ingrid J Poulton
  5. Tsuyoshi Isojima
  6. Jonathan H Gooi
  7. T John Martin
  8. Natalie A Sims
(2020)
Cortical bone maturation in mice requires SOCS3 suppression of gp130/STAT3 signalling in osteocytes
eLife 9:e56666.
https://doi.org/10.7554/eLife.56666
  2. Download BibTeX
  3. Download .RIS
7 figures, 2 videos, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Download asset Open asset
Transition to compact and highly mineralised bone is delayed in Dmp1Cre:Socs3f/f mice until longitudinal growth has ceased.

(A) Schematic for studying time course of corticalisation in female Dmp1Cre:Socs3f/f mice. (B) Femur length at 6, and 12 to 16 weeks of age in female Dmp1Cre:Socs3f/f and control mice; *p<0.05 for …

Figure 1—source data 1

Raw data for cortical porosity measurements.

https://cdn.elifesciences.org/articles/56666/elife-56666-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Download asset Open asset
Cortical porosity of the femoral diaphysis of Dmp1Cre and Dmp1Cre:Socs3f/f mice at 12 weeks of age.

Data are mean ± SEM; n = 11 mice per group; p value derived by Welch’s t-test.

Figure 2
Download asset Open asset
Transition to more highly mineralised bone during maturation of the metaphysis in female control (Dmp1Cre) and Dmp1Cre:Socs3f/f femora.

(A) Diagram showing region measured, and Otsu thresholding of a raw micro-CT image. (B) Bone volume, as a percentage of total metaphyseal volume, segregated by low, mid and high density volumes, at …

Figure 2—source data 1

Raw data for bone density measurements at all three densities.

https://cdn.elifesciences.org/articles/56666/elife-56666-fig2-data1-v1.xlsx
Figure 3 with 3 supplements
Download asset Open asset
Bone becomes more mineralized with age, and with increasing distance from the growth plate in Dmp1Cre mice; this is delayed in Dmp1Cre: Socs3f/f mice.

(A) The metaphyseal region was analysed in consecutive 9 micron slices. (B–E) Bone volume at low-, mid-, and high-density mineral levels in each slice from the distal to proximal end of the …

Figure 3—source data 1

Bone volume at low-, mid-, and high-density mineral levels in each slice from the distal to proximal end of the metaphyseal region at 12 weeks and 15 weeks in Dmp1Cre and Dmp1Cre:Socs3f/f mice.

https://cdn.elifesciences.org/articles/56666/elife-56666-fig3-data1-v1.xls
Figure 3—figure supplement 1
Download asset Open asset
Total bone area of the metaphyseal slices in Dmp1Cre and Dmp1Cre:Socs3f/f mice at 12 and 15 weeks of age (B,D,F).

Data are mean ± SEM; n = 9–11 mice per group.

Figure 3—figure supplement 2
Download asset Open asset
Comparison of high density, (A,B), mid-density (C,D) and low density (E,F) bone between Dmp1Cre and Dmp1Cre:Socs3f/f mice at 12 (A,C,E) and 15 weeks of age (B,D,F).

Data are mean with SEM shown by dashed lines, taken slice by slice through the metaphysis (see Figure 3A for schematic). Gray region indicates significant difference between genotypes by repeated …

Figure 3—figure supplement 3
Download asset Open asset
Direct comparison of archived scans from Dmp1Cre and Dmp1Cre:Socs3f/f mice at 26 weeks of age.

(A) Bone volume, as a percentage of total metaphyseal volume, segregated by low, mid and high density volumes; changes in low-, mid- and high-density bone are indicated by coloured asterisks; error …

Figure 4
Download asset Open asset
STAT3 phosphorylation response is magnified and extended in calvarial bone from male Socs3f/f (control) and Dmp1Cre:Socs3f/f mice treated with gp130-dependent stimuli (A-C), and elevated under basal conditions in Dmp1Cre:Socs3f/f osteocytes (D).

(A-C) Mice were given a single calvarial injection of murine oncostatin M (mOSM) (A), murine leukaemia inhibitory factor (mLIF) (B), or human interleukin- 11 (hIL-11) (C). Calvariae were collected …

Figure 4—source data 1

Uncropped gels for phospho-STAT3 (left panels) and total STAT3/pan actin (right panels) for calvarial samples from Socs3f/f and Dmp1Cre:Socs3f/f mice treated with OSM (A), LIF (B) or IL-11 (C).

Details of molecular weight markers and legends for the gels are in Figure 4.

https://cdn.elifesciences.org/articles/56666/elife-56666-fig4-data1-v1.pdf
Figure 5 with 1 supplement
Download asset Open asset
Rescue of delayed corticalisation in 12 week old male and female Dmp1Cre:Socs3f/f:Il6stf/f mice.

Metaphyseal cortical porosity in female and male control (Dmp1Cre) and Dmp1Cre:Il6stf/f mice (A) and Dmp1Cre:Socs3f/f and Dmp1Cre:Socs3f/f:Il6stf/f mice (B) and representative Otsu-based …

Figure 5—source data 1

Bone volume at low-, mid-, and high-density mineral levels in each slices from the distal to proximal end of the metaphyseal region in 12 week old female and male Dmp1Cre:Socs3f/f mice and Dmp1Cre:Socs3f/f:Il6stf/f mice.

https://cdn.elifesciences.org/articles/56666/elife-56666-fig5-data1-v1.xls
Figure 5—figure supplement 1
Download asset Open asset
Direct comparison of high density (A,B), mid-density (C,D) and low-density (E,F) bone between Dmp1Cre:Socs3f/f and Dmp1Cre:Socs3f/f:Il6stf/f mice.

Data are mean with SEM shown by dashed lines, taken slice by slice through the metaphysis (see Figure 3A for schematic). Data are mean ± SEM; n = 9–11 mice per group. Gray region indicates …

Figure 6 with 1 supplement
Download asset Open asset
Closure of cortical pores in Dmp1Cre:Socs3f/f mice by Il6st knockdown involves reduced cortical resorption and reduced STAT3 phosphorylation.

(A,B) Representative calcein labelling in Dmp1Cre:Socs3f/f and Dmp1Cre:Socs3f/f:Il6stf/f mice. Pink dashed lines show transcortical pores. (C–E) Histomorphometry of cortical void area (Vd.Ar/Ct.Ar), …

Figure 6—figure supplement 1
Download asset Open asset
Additional histological images, including analysis of calvarial bone.

(A) Ploton silver stain in the tibial lateral metaphysis of a 12 week old Dmp1Cre mouse; for comparison with Figure 6M,N. (B) Area of voids (diplöe) as a proportion of calvarial bone from 12 week …

Figure 7
Download asset Open asset
Model of cortical maturation and the role of SOCS3 in osteocytes.

(A) During longitudinal growth, trabeculae arising from remodelling of the growth plate consolidate, initially forming porous woven cortical bone with irregular oriented osteocytes with rounded cell …

Videos

Video 1
Download asset
Video showing the sequence of metaphyseal images obtained by micro-CT in control (Dmp1Cre) mice from 12 to 15 weeks of age.

Pseudocolourisation reflects the same density scale shown in Figure 1.

Video 2
Download asset
Video showing the sequence of metaphyseal images obtained by micro-CT in Dmp1Cre:Socs3f/f mice from 12 to 15 weeks of age.

Pseudocolourisation reflects the same density scale shown in Figure 1.

Tables

Table 1
Phenotypic data from 12 week old Dmp1Cre:Il6stf/f mice, including mRNA levels of the targeted gene in flushed femoral samples (Il6st:B2m), femoral trabecular and cortical structure (by micro-computed tomography) and tibial trabecular histomorphometry in the secondary spongiosa.

Data are mean ± SEM. *, p<0.05, **, p<0.01; ***, p<0.001 vs sex- and age-matched controls (Dmp1Cre) by two-way ANOVA with Šidák post-hoc test.

Parametermalefemale
Dmp1CreDmp1Cre:Il6stf/fDmp1CreDmp1Cre:Il6stf/f
Number of samples71066
IL6st:B2m (mRNA)1.14 ± 0.210.52 ± 0.08**0.88 ± 0.150.40 ± 0.18
Trabecular bone volume (%)
Trabecular number (/mm)
Trabecular Thickness (μm)
Trabecular Separation (μm)		13.72 ± 1.26
2.54 ± 0.21
53.5 ± 0.8
217 ± 14.01		8.24 ± 0.96**
1.27 ± 0.12***
64.1 ± 2.6***
469 ± 26***		10.82 ± 1.06
2.38 ± 0.22
45.6 ± 1.0
239 ± 15		6.06 ± 0.51**
1.12 ± 0.08***
54.0 ± 1.3***
415 ± 29***
Periosteal Perimeter (mm)
Marrow Area (mm2)
Moment of Inertia (mm4)		7.21 ± 0.14
1.10 ± 0.04
0.395 ± 0.021		8.14 ± 0.25***
1.37 ± 0.08***
0.566 ± 0.51***		6.78 ± 0.13
0.94 ± 0.04
0.344 ± 0.02		7.29 ± 0.10*
1.11 ± 0.02*
0.404 ± 0.009
Bone Formation Rate/Bone Surface (%)
Double labelled Surface/Bone Surface (%)
Mineral Appositional Rate (μm/day)
Osteoclast Surface/Bone Surface (%)		0.454 ± 0.019
29.1 ± 1.8
1.30 ± 0.08
4.39 ± 0.58		0.295 ± 0.048**
20.1 ± 2.8**
1.10 ± 0.14
6.59 ± 0.89		0.704 ± 0.03
26.6 ± 1.5
1.98 ± 0.24
3.61 ± 0.45		0.481 ± 0.065*
15.1 ± 1.8**
1.63 ± 0.19
4.82 ± 0.83
Table 2
mRNA analysis of osteoclast-related genes in flushed femora from 12 week old Dmp1Cre:Socs3f/f and Dmp1Cre:Socs3f/f: Il6stf/f.

Data are mean ± SEM normalised to the geometric mean of B2m and Hprt1; p values from two-way ANOVA analysis are shown in the right column, including p values for the comparison of male vs female, Dmp…

Parametermalefemalep values
Dmp1Cre:Socs3f/fDmp1Cre:Socs3f/f:Il6stf/fDmp1Cre:Socs3f/fDmp1Cre:Socs3f/f:Il6stf/f
Number of samples108108
Tnfsf11 (RANKL)


Tnfrsf11b (OPG)


Acp5 (TRAP)		0.015 ± 0.002


0.067 ± 0.014


3.64 ± 0.36		0.016 ± 0.004


0.097 ± 0.017


1.94 ± 0.26		0.015 ± 0.003


0.026 ± 0.004


10.25 ± 1.45		0.015 ± 0.003


0.044 ± 0.009


7.41 ± 1.12		Male vs female: 0.752;
Genotype: 0.841;
Interaction: 0.923.
Male vs female: 0.0003;
Genotype: 0.048;
Interaction: 0.635.
Male vs female:<0.0001;
Genotype: 0.028;
Interaction: 0.568.
Table 3
Results of three point bending tests of femora from male and female 12 week old Dmp1Cre:Socs3f/f and Dmp1Cre:Socs3f/f:Il6stf/f mice.

Data is mean ± SEM. *, p<0.05; **, p<0.01 vs Dmp1Cre:Socs3f/f by two-way ANOVA with Šidák post-hoc test.

Parametermalefemale
Dmp1Cre:Socs3f/fDmp1Cre:Socs3f/f:Il6stf/fDmp1Cre:Socs3f/fDmp1Cre:Socs3f/f:Il6stf/f
Number of samples911109
Ultimate Force (N)
Ultimate Deformation (mm)
Yield Force (N)
Yield Deformation (mm)
Post-Yield Deformation (mm)
Energy to Failure (mJ)
Ultimate Stress (MPa)
Ultimate Strain (%)
Yield Stress (MPa)
Yield Strain (%)
Post-Yield Strain (%)		16.48 ± 0.69
0.304 ± 0.025
15.44 ± 1.75
0.240 ± 0.022
0.063 ± 0.018
2.61 ± 0.31
47.30 ± 4.69
0.033 ± 0.003
41.65 ± 5.20
0.026 ± 0.003
0.007 ± 0.002		15.31 ± 0.60
0.330 ± 0.02
11.29 ± 0.70**
0.212 ± 0.022
0.126 ± 0.019*
2.94 ± 0.25
39.67 ± 1.72
0.036 ± 0.003
28.97 ± 1.46*
0.023 ± 0.003
0.014 ± 0.002*		13.40 ± 0.25
0.350 ± 0.029
11.03 ± 0.58
0.282 ± 0.024
0.068 ± 0.013
2.34 ± 0.16
63.96 ± 2.39
0.034 ± 0.003
52.46 ± 3.02
0.028 ± 0.002
0.007 ± 0.001		13.79 ± 0.59
0.350 ± 0.019
10.19 ± 0.69
0.219 ± 0.026
0.152 ± 0.020**
2.99 ± 0.19*
54.29 ± 2.33
0.037 ± 0.002
40.42 ± 3.44
0.023 ± 0.003
0.016 ± 0.002**
Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Strain, strain background (Mus musculus)Dmp1CreLynda Bonewald (Indiana University, USA)Tg(Dmp1-cre)1JqfeC57BL/6 background
Strain, strain background (Mus musculus)Socs3tm1WsaWarren Alexander (Walter and Eliza Hal Institute)Socs3tm1WsaC57BL/6 background
Strain, strain background (Mus musculus)Il6st-flox mouse strainNCRR-NIH-supported KOMP RepositoryIL6sttm1a(KOMP)MbpC57BL/6 background
Antibodyphospho-STAT3 (Rabbit polyclonal)Cell SignallingCat #9131
RRID:AB_331586		IHC (1:100), WB (1:400)
AntibodySTAT3
(mouse monoclonal)		Cell SignallingCat#9139
RRID:AB_331757		WB (1:400)
AntibodyGoat anti-MouseDakoCat#P0447
RRID:AB_2617137		WB (1:1500)
AntibodySwine anti-RabbitDakoCat#P0217 RRID:AB_2728719IHC (1:250)
WB (1:2000)
Peptide, recombinant proteinMurine oncostatin MR and D SystemsCat#495-MO-025
Peptide, recombinant proteinhuman IL-11R and D SystemsCat#218-IL-025
Peptide, recombinant proteinmurine LIFMerck Millipore#LIF2010
Peptide,
recombinant protein		Murine LeptinR and D Systems#498-OB-01M
Peptide, recombinant proteinmurine G-CSFR and D Systems#414-CS-005

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/56666/elife-56666-transrepform-v1.docx

Download links