R-spondin 3 deletion induces Erk phosphorylation to enhance Wnt signaling and promote bone formation in the appendicular skeleton
- School of Dental Medicine, Harvard University, United States
- German Cancer Research Center, DKFZ-ZMBH Alliance, Germany
- Institute of Molecular Biology (IMB), Germany
- Department of Medicine, Harvard Medical School, United States
- Endocrine Unit, Massachusetts General Hospital, United States
Figures
Figure 1 with 3 supplements
Skeletal phenotype of mice with Rspo3 haplo-insufficiency.
(a) Rspo3 expression in marrow depleted long bones, isolated from WT and Rspo3+/- mice (n=4–5). Data show all samples and are the mean ± SEM **p<0.005 by unpaired Student’s t-test. (b) Histomorphometric analysis in 6, 12, and 18 wk males and females (red line = WT, black line = Rspo3+/-). (n=3–9). Data are the mean ± SEM. Two-way ANOVA followed by Fisher’s LSD test. *p<0.05, **p<0.005, ****p<0.0001. (c) Representative images of Von Kossa staining in 12 wk-old WT and Rspo3+/- male tibiae. (n=8-9). Scale bars = 1.0 mm. (d) Histomorphometric analysis of 12 wk-old WT and Rspo3+/- female and male tibiae (n=3–9). Data show all samples and the mean ± SEM. *=p<0.05, **p<0.005 by unpaired Student’s t-test. Red circles = WT, Black circles = Rspo3+/-. (e) Representative images of double labeling in trabecular bone in 12 wk-old WT and Rspo3+/- male tibiae. (n=8-9). Scale bars = 10 μm.
Figure 1—figure supplement 1
Skeletal phenotype of mice with Rspo3 haploinsufficiency.
(a) Rspo3 expression in calvarial OBs Data show all samples and the mean ± SEM (n=8) .*p<0.05 compared with WT by unpaired Student’s t-test. (b) Representative skeletal preparations of WT and Rspo3+/- newborns ( n=3–4). (c) Representative images of Von Kossa staining of 6 and 18 wk old WT and Rspo3+/- males. (n=6-8). Scale Bars=1.0mm.
Figure 1—figure supplement 2
Axial skeletal phenotype of Rspo3+/- mice.
(a) Representative images of Von Kossa staining in 12 wk old WT and Rspo3+/- L5 vertebrae. (n=7-9). Scale bars=500μm. (b) Structural parameters by μCT analysis of WT and Rspo3+/- females and males (n=7–9). (c) BV/TV, Tb.N, MAR, BFR/BS, N.OC/BPm and N.Ob/BPm by histomorphometric analysis in WT and Rspo3+/- females and males (n=6–7). Data show all samples and the mean ± SEM. Red circles = WT and black circles = Rspo3+/-. No significant difference by Student's T-test.
Figure 1—figure supplement 3
Rspo3 haploinsufficiency does not affect osteoclastogenesis.
(a) Rspo3 expression in BMM-derived osteoclasts. Data show all samples and the mean ± SEM (n=3) **p = 0.0019 by unpaired Student's t-test. (b) Representative images of TRAP staining of WT and Rspo3+/- BMM-derived osteoclasts. (n=3). Scale bars = 100μm (c) Nfatc1, Trap, and Ctsk expression in WT and Rspo3+/- BMM-derived osteoclasts Data show all samples and the mean ± SEM. (n=3). No significant difference by unpaired Student's T-test. (d) Representative mix-matched experiment and quantification of TRAP+ BMM-derived osteoclasts. Data show the mean ± SEM (n=3). No significant difference by Two-way ANOVA followed by Fisher's LSD test.
Figure 2
Rspo3 haplo-insufficiency increases the % of osteoprogenitors.
(a) Representative images of Flow citometry analysis. (b) Quantification of the % of Lin-Cd45-Cd31-CD51+Sca+ cells in WT and Rspo3+/- bone marrow. Data show all samples and the ± SEM (n=10) *=p< 0.05 by unpaired Student’s t-test. (c) Representative images of CFU-F and CFU-OB assay and quantification in WT and Rspo3+/- mice treated in the absence and presence of Rspo3. Data show all samples and the mean ± SEM (n=4–9) *=p<0.05, **=p<0.005 by two-way ANOVA followed by Fisher’s LSD test.
Figure 3 with 1 supplement
Appendicular skeletal phenotype of mice with Rspo3 targeted deletion in Runx2+ cells (Rspo3-OB-cKO).
(a) Rspo3 expression in marrow depleted long bones, showing deletion efficiency (n=6–7). Data show all samples and are the mean ± SEM. **p=0.0019 by unpaired Student’s t-test. (b) Representative images of Von Kossa staining in 8 wk old Rspo3fl and Rspo3-OB-cKO tibiae. Scale bars = 200 μm (c) BV/TV, MAR, BFR/BS, and N.Ob/BPm by histomorphometric analysis in Rspo3fl and Rspo3-OB-cKO females and males (n=7–10). (d) Structural parameters by μCT analysis Rspo3fl and Rspo3-OB-cKO femur (n=6–7). Data show all samples and the mean ± SEM *=p<0.05, **=p<0.005, ****=p<0.0001 compared with the correspondent Rspo3fl group by unpaired Student’s t-test. Open circles = Rspo3 fl and filled circles = Rspo3-OB-cKO.
Figure 3—figure supplement 1
Axial (L5) and appendicular (femur) skeletal phenotype of Runx2-Cre, WT and Rspo3fl mice.
(a) Structural parameters by μCT analysis at 12 weeks of age (n=3–6). Data show all samples and the mean ± SEM. square = Runx2 Cre, triangle = WT and black circles = Rspo3 fl.
Figure 4
Axial skeletal phenotype of mice with Rspo3 targeted deletion in Runx2+ cells (Rspo3-OB-cKO).
(a) Rspo3 expression in vertebrae, showing deletion efficiency (n=3–5). Data show all samples and are the mean ± SEM **p=0.005 by unpaired Student’s t-test. (b) Structural parameters by μCT analysis Rspo3fl and Rspo3-OB-cKO L5 vertebrae (n=7–10). (c) Representative images of Von Kossa staining in 8 wk old Rspo3fl and Rspo3-OB-cKO L5 vertebrae. (n=7-10). Scale bars = 500 μm (d) BV/TV, Tb.N, MAR, BFR/BS, N.OC/BPm, and N.Ob/BPm by histomorphometric analysis in Rspo3fl and Rspo3-OB-cKO females and males (n=7–10). Data show all samples and the mean ± SEM *=p<0.05, **=p<0.005, ****=p<0.0001 compared with the correspondent Rspo3fl group by unpaired Student’s t-test. Open circles = Rspo3 fl and filled circles = Rspo3-OB-cKO.
Figure 5
Rspo3 haplo-insufficiency leads to Wnt signaling activation.
(a) Expression of Rspo3, Runx2 and selected Wnt target genes in BMSCs (n=3–4). Data show all samples and the mean ± SEM. (b) Representative images and quantification of active β-catenin by Western analysis in BMSC isolated from WT and Rspo3+/- mice (n=7). (c) Expression of selected Wnt target genes in marrow depleted long bones (n=3–7). Data show all samples and the mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005 by unpaired Student’s t-test.
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Figure 5—source data 1
Rspo3 haplo-insufficiency leads to Wnt signaling activation.
Representative image active β-catenin by Western analysis in BMSC isolated from WT and Rspo3+/- mice (n=7).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig5-data1-v2.zip
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Figure 5—source data 2
Rspo3 haplo-insufficiency leads to Wnt signaling activation.
Representative image of actin by Western analysis in BMSC isolated from WT and Rspo3+/- mice (n=7).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig5-data2-v2.zip
Figure 6 with 1 supplement
Rspo3 deletion leads to Wnt signaling activation in vitro.
(a) Luciferase assay and Wnt target gene expression in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=6–11). Data show all samples and the mean ± SEM. (b) Representative images and quantification of active β−catenin, pLrp6 and Tcf1 by Western analysis in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=3–7). Data show all samples and the mean ± SEM. a=p<0.05 vs vehicle WT, b=p<0.05 vs Wnt3a–treated WT and c=p<0.05 vs Wnt3a treated Rspo3-/- by two-way ANOVA followed by Fisher’s LSD test.
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Figure 6—source data 1
Rspo3 deletion leads to Wnt signaling activation in vitro.
Representative image of active β−catenin by Western analysis in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=7).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig6-data1-v2.zip
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Figure 6—source data 2
Rspo3 deletion leads to Wnt signaling activation in vitro.
Representative image of pLrp6 by Western analysis in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=7).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig6-data2-v2.zip
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Figure 6—source data 3
Rspo3 deletion leads to Wnt signaling activation in vitro.
Representative image of actin by Western analysis in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=7).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig6-data3-v2.zip
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Figure 6—source data 4
Rspo3 deletion leads to Wnt signaling activation in vitro.
Representative image of Tcf1 by Western analysis in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=3).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig6-data4-v2.zip
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Figure 6—source data 5
Rspo3 deletion leads to Wnt signaling activation in vitro.
Representative image of Gapdh by Western analysis in WT and Rspo-/- MEFs treated w/wo Wnt3a (n=3).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig6-data5-v2.zip
Figure 6—figure supplement 1
Wnt3a and Rspo3 treatment in WT MEFs.
(a) Luciferase assay in WT MEFs treated w/wo Wnt3a and Rspo3 (n=6–7). Data show all samples and the mean ± SEM, a=p<0.05 vs vehicle WT, b=p<0.05 vs Rspo3 treated WT and c=p<0.05 vs Wnt3a treated WT by two-way ANOVA followed by Fisher’s LSD test. (b) Expression of Rspo3 in WT and Rspo3-/- MEFS (n=7). Data show all samples and the mean ± SEM. ****p<0.0001 by unpaired Student’s t-test.
Figure 7
Rspo3 deletion/ haplo-insufficiency impairs Dkk1 efficacy.
(a) Regulation of Rspo3 by Wnt3a and Dkk1 in WT MEFs (n=3) Data are the mean ± SEM. ***p<0.0005, ****p<0.0001, by Student’s t-test. (b) Luciferase assay in WT and Rspo-/- MEFs treated w/wo Wnt3a and increasing doses of Dkk1 (n=3–4). Data are the mean ± SEM **p<0.005, ***<p< 0.0005 compared with vehicle same genotype by unpaired Student’s t-test. (c) Representative images and quantification of active β-catenin and pLrp6 by Western analysis in WT and Rspo3-/- MEFs treated w/wo Wnt3a and increasing doses of Dkk1 (n=3). Data are the mean ± SEM *p<0.05, **p<0.005 vs WT vehicle, # p<0.05, vs Wnt3a–treated same genotype by unpaired Student’s t-test. (d) Representative images of Von Kossa staining in 6 wk old female mice (n=5-6). Scale bars = 1.0 mm. (e) BV/TV, MAR, BFR/BS, and N.Ob./B.pm by histomorphometric analysis in females (n=5–6). Data show all samples and the mean ± SEM a=p<0.05 compared with control mice, b=p < 0.05 compared with Rspo3+/- mice, c=p<0.05 compared with Dkk1-Tg mice by two-way ANOVA followed by Fisher’s LSD test.
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Figure 7—source data 1
Rspo3 deletion/ haplo-insufficiency impairs Dkk1 efficacy.
Representative image of active β-catenin and pLrp6 by Western analysis in WT and Rspo3-/- MEFs treated w/wo Wnt3a and increasing doses of Dkk1 (n=3).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig7-data1-v2.zip
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Figure 7—source data 2
Rspo3 deletion/ haplo-insufficiency impairs Dkk1 efficacy.
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig7-data2-v2.zip
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Figure 7—source data 3
Rspo3 deletion/ haplo-insufficiency impairs Dkk1 efficacy.
Representative image of actin by Western analysis in WT and Rspo3-/- MEFs treated w/wo Wnt3a and increasing doses of Dkk1 (n=3).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig7-data3-v2.zip
Figure 8
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
(a) Representative images and quantification of pERK, active β-catenin and pLrp6 levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126. (b) Expression of Wnt target genes in WT and Rspo3-/- MEFs treated w/wo Wnt3a and U0126. Data show all samples and the mean ± SEM (n=3–4) *p<0.05, **p<0.005 vs vehicle of the same genotype, ^=p<0.05 vs WT vehicle and # p<0.05 vs Wnt3a–treated same genotype by unpaired Student’s t-test.
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Figure 8—source data 1
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of active β-catenin in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data1-v2.zip
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Figure 8—source data 2
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of tubulin levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data2-v2.zip
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Figure 8—source data 3
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of pERK levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data3-v2.zip
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Figure 8—source data 4
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of total ERK levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data4-v2.zip
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Figure 8—source data 5
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of pLrp6 levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data5-v2.zip
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Figure 8—source data 6
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of total Lrp6 levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data6-v2.zip
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Figure 8—source data 7
Erk signaling is involved in the Wnt signaling activation seen in the absence of Rspo3.
Representative image of Gapdh levels by western analysis in WT and Rspo3-/- MEFs treated w/wo w/wo Wnt3a and U0126 (n=3–4).
- https://cdn.elifesciences.org/articles/84171/elife-84171-fig8-data7-v2.zip
Figure 9
Effect of Erk signaling inhibition of CFU assays.
Representative images of CFU-F and CFU-OB and quantification in WT and Rspo3+/- mice treated with and w/o U0126. Data show all samples and the mean ± SEM (n=3) *=p<0.05, **=p<0.005, ****=p<0.0001 two-way ANOVA followed by Fisher’s LSD test.
Figure 10
Proposed model.
Rspo3 has a dual mode of action to regulate canonical Wnt signaling and thereby bone formation. This duality is based on the regulation of two distinct signaling cascades and their crosstalk: Rspo3 functions via both the Lgr/Rnf43/Znrf3 and the Lgr/Erk axes. In the presence of Rspo3, the Rspo3/Lgr/Rnf43/Znrf3 axis boosts Wnt signaling strengths by the membrane clearance of Rnf43/Znrf3 and subsequent stabilization of Fzd receptors. In addition, binding of Rspo3 to Lgr impairs Erk signaling likely due to the membrane clearance of the Lgr/Rnf43/Znrf3 receptors, preventing Erk signaling activation. Deletion of Rspo3 would dampen Wnt signaling at the cell surface by preventing the Rnf43/Znrf3 effects while promoting Erk activation downstream of Lgr receptors in turn enhancing Lrp5/6 phosphorylation and β−catenin stabilization intracellularly, which has a more potent effect and overcompensates the decrease in Rspo3-dependent proximal Wnt activation in osteoblasts and their progenitors. Figure created with Biorender.
Tables
Table 1
Histomorphometric analysis of WT and Rspo3+/- females.
| Parameters | 6 wk | 12 wk | 18 wk | Two way ANOVA | |||||
|---|---|---|---|---|---|---|---|---|---|
| WT(n=6) | Rspo3+/-(n=7) | WT(n=4) | Rspo3+/-(n=4) | WT(n=8) | Rspo3+/-(n=6) | Genotype | Age | Interaction | |
| BV/TV (%) | 7.24±0.98 | 8.8±0.88 | 5.84±1.01 | 12.2±0.72*** | 3.28±0.51 | 6.06±1.1* | <0.0001 | <0.0001 | NS |
| Tb.Th (μm) | 28.3±0.91 | 33.6±1.5* | 35.8±2.54 | 40.7±1.31 | 28.2±1.55 | 36.8±2.7** | 0.0003 | 0.0032 | NS |
| Tb.N (/mm) | 2.53±0.27 | 2.6±0.20 | 1.63±0.26 | 3.00±0.15** | 1.13±0.16 | 1.61±0.25 | 0.0025 | <0.0001 | 0.0429 |
| Tb.Sp (μm) | 397±52.6 | 368±26.4 | 618±86.8 | 297±17.1* | 1050±192 | 690±133 | 0.0378 | 0.0008 | NS |
| MAR (μm/day) | 1.4±0.11 | 1.8±0.06 | 1.39±0.17 | 2.5±0.14*** | 1.08±0.15 | 1.5±0.20* | <0.0001 | 0.0016 | NS |
| MS/BS (%) | 22.2±2.06 | 31.4±4.05* | 23.0±3.25 | 27.8±1.63 | 26.8±1.90 | 27.3±1.55 | 0.0394 | NS | NS |
| BFR/BS (μm3/μm2/year) | 116.2±16.6 | 207±30.4** | 116±16.9 | 251±6.43** | 110±17.6 | 153±25.1 | <0.0001 | NS | NS |
| N.Ob/B.Pm (/mm) | 6.72±0.79 | 6.85±0.64 | 3.83±0.45 | 13±1.94**** | 6.60±0.39 | 8.4±0.69 | <0.0001 | NS | 0.0001 |
| Ob.S/B.Pm (%) | 10.3±1.20 | 10.5±1.44 | 4.89±0.38 | 16.3±2.2**** | 9.66±0.52 | 12.3±0.53 | <0.0001 | NS | 0.0007 |
| OS/BS (%) | 4.68±0.78 | 5.51±1.23 | 2.51±0.30 | 9.17±0.77*** | 4.84±0.66 | 8.3±0.74** | <0.0001 | NS | 0.0263 |
| O.Th (μm) | 3.89±0.30 | 4.64±0.37 | 2.76±0.13 | 4.36±0.09* | 2.78±0.37 | 4.24±0.3** | 0.0003 | NS | NS |
| N.Oc/B.Pm (/mm) | 1.08±0.13 | 0.99±0.11 | 3.64±0.31 | 3.67±0.14 | 2.01±0.26 | 2±0.48 | NS | <0.0001 | NS |
| Oc.S/B.Pm (%) | 2.98±0.40 | 3.07±0.37 | 7.89±0.42 | 8.77±0.55 | 5.85±0.86 | 5.7±1.17 | NS | <0.0001 | NS |
| ES/BS (%) | 4.41±1.06 | 4.05±0.42 | 1.67±0.48 | 2.96±0.43 | 6.85±0.82 | 6.74±1.28 | NS | <0.0001 | NS |
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Data are expressed as Mean ± SEM. Two way ANOVA followed by Fisher’s LSD post-hoc test. *p<0.05, **=p < 0.005, ***=p < 0.001, ****=p < 0.0001 compared with age-matched WT females.
Additional files
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Supplementary file 1
Histomorphometric analysis of WT and Rspo3+/- males.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp1-v2.docx
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Supplementary file 2
Histomorphometric analysis of the tibia midshaft in 12 wk-old WT and Rspo3+/- females.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp2-v2.docx
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Supplementary file 3
Histomorphometric analysis of the tibia midshaft in 12 wk-old WT and Rspo3+/- males.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp3-v2.docx
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Supplementary file 4
Histomorphometric analysis of 12 wk-old WT and Rspo3+/- vertebrae.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp4-v2.docx
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Supplementary file 5
Histomorphometric analysis of 8 wk-old Rspo3fl and Rspo3-OB-cKO mice.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp5-v2.docx
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Supplementary file 6
Histomorphometric analysis of 8 wk-old Rspo3fl and Rspo3-OB-cKO vertebrae.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp6-v2.docx
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Supplementary file 7
Histomorphometric analysis of Control, Rspo3+/-,Dkk1-Tg and Rspo3+/-; Dkk1-Tg female mice at 6 wk of age.
- https://cdn.elifesciences.org/articles/84171/elife-84171-supp7-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84171/elife-84171-mdarchecklist1-v2.pdf
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R-spondin 3 deletion induces Erk phosphorylation to enhance Wnt signaling and promote bone formation in the appendicular skeleton
eLife 11:e84171.
https://doi.org/10.7554/eLife.84171
