Osteoclast-secreted SLIT3 coordinates bone resorption and formation

Abstract

Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.

Keywords: Bone Biology; Coupling factors.

Figure 1. Increased SLIT3 production during osteoclast differentiation.

(A) SLIT3 concentrations measured by ELISA in the CM from lineages of osteoclasts (OCs) and osteoblasts (OBs). The levels were normalized by the protein amount of each cell lysate. (B) Quantitative RT-PCR to measure Slit3 levels in RAW264.7 cells. Cells were treated with RANKL (15 ng/ml) for 4 days to induce osteoclast-like cells. Quantitative gene expression analysis was performed by RT-PCR using the LightCycler 480 system. 18S rRNA was used as an internal control. Ratios of Slit3 and 18S rRNA expression levels were calculated using the 2^–ΔΔCT method. (C and D) Quantitative RT-PCR and luciferase assays of Slit3 expression and Slit3 promoter activity, respectively, after transfection with 50 ng cDNAs expressing Creb or NF-κB subunits p50 or p65 for 48 hours, in RAW264.7 cells. The values were normalized to the 18S rRNA level and β-galactosidase activity, respectively. (E and F) Quantitative RT-PCR and luciferase assays before and after pretreatment with inhibitors of NF-κB p50 and CREB (PDTC and KG501, respectively) in BMMs with M-CSF. (G and H) ChIP assay after IP with antibodies against NF-κB p50 and phosphorylated CREB in RAW264.7 cells to assess the activation of these factors at Slit3 promoter regions. RANKL (100 ng/ml) treatment duration was 1 hour. Data are presented as mean ± SEM of 3–4 independent experiments. *P < 0.05 vs. untreated or empty vector–transfected control using the Mann-Whitney U test or Kruskal-Wallis test followed by Bonferroni’s correction.

Figure 2. SLIT3 stimulates osteoblast migration and proliferation.

(A) Quantitative RT-PCR and Western blot of SLIT3 after transfection with Slit3 siRNA for 24 hours in mouse mature osteoclasts. Mouse BMMs were differentiated into mature osteoclasts with 15 ng/ml RANKL and 15 ng/ml M-CSF for 3 days, and CM were collected during the subsequent 24 hours with or without Slit3 siRNA. Directional migration of MC3T3-E1 cells was assessed after treatment with the collected CM for 24 hours. (B and C) Directional migration (B) and proliferation (C) of mouse calvaria osteoblasts with SLIT3 for 24 hours and 48 hours, respectively. (D) Intrabone marrow mobilization of GFP-labeled MC3T3-E1 cells (n = 5 per group). (E) Western blot of β-catenin after IP with N-cadherin in mouse calvaria osteoblasts with 1.0 μg/ml SLIT3 for 60 minutes. The experiment was performed without WNTs. (F) TCF/LEF reporter assay with 1.0 μg/ml SLIT3 for 48 hours in MC3T3-E1 cells. (G) Quantitative RT-PCR and Western blot of β-catenin after transfection with β-catenin siRNA (Ctnnb1) for 24 hours in mouse calvaria osteoblasts. Directional migration and proliferation were assessed after treatment with 1.0 μg/ml SLIT3 for 24 hours and 48 hours, respectively. (H) Von Kossa staining of femur (upper) and lumbar spine (lower) of 7-week-old male Slit3^–/– mice and WT littermates (n = 4–5 per group). Trabecular bone parameters in the femur were assessed by histomorphometric analyses. BV/TV, bone volume/tissue volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. Scale bars: 500 μm. Detailed information appears in the Supplemental Methods. Data are presented as mean ± SEM. In vitro experiments were performed 3–5 times independently. *P < 0.05 vs. untreated control or WT mice using the Mann-Whitney U test or Kruskal-Wallis test followed by Bonferroni’s correction.

Figure 3. Regulation of bone resorption by SLIT3.

(A) Histomorphometric analyses including calcein double labeling of the femur of 7-week-old male Slit3^–/– mice and WT littermates (n = 4–5 per group). BFR/BS, bone formation rate per bone surface; MAR, mineral apposition rate; N.Ob/BS, osteoblast number/bone surface; ES/BS, eroded surface/bone surface; OC/BS, multinucleated osteoclast number/bone surface. Scale bars: 10 μm. (B) Serum bone turnover markers in 7-week-old male Slit3^–/– mice and WT littermates (n = 9–10 per group). (C) TRAP staining of mouse BMMs with 15 ng/ml M-CSF and 15 ng/ml RANKL for 4 days. (D) The same methods were performed in BMMs obtained from 6-week-old male or female Slit3^–/– mice and WT littermates (n = 3 per group). (E) Semiquantitative RT-PCR of Trap, Ctr, and Dc-stamp in mouse BMMs with M-CSF and RANKL. (F) TRAP staining of mouse BMMs with M-CSF and RANKL for 2–3 days. The nuclei number per TRAP-positive cell was counted. (G) Intrabone marrow mobilization of red fluorescent protein–labeled (RFP-labeled) BMMs (n = 5 per group). (H) Western blot of RhoA, Rac, and Cdc42 following 1.0 μg/ml SLIT3 treatment for 15 minutes in mouse BMMs with M-CSF and RANKL. (I) Western blot of Rac GTPase after transfection with empty vector (pCMV5) or mutationally activated Rac1 (Rac1-V12) in mouse BMMs. TRAP staining was also performed at 4 days after transfection. (J) Directional migration of mouse BMMs with 1.0 μg/ml SLIT3 for 24 hours after transfection. Detailed information is supplied in the Supplemental Methods. Data are presented as mean ± SEM. In vitro experiments were performed 3–4 times independently. *P < 0.05 vs. untreated or empty vector–transfected control or between indicated groups using the Mann-Whitney U test or Kruskal-Wallis test followed by Bonferroni’s correction.

Figure 4. Osteopenic phenotypes in Robo1^–/– mice.

(A) Expression of Robo mRNA in mouse calvaria osteoblasts using semiquantitative RT-PCR. Mouse brain and vascular tissues were used as positive controls (+) for Robo1–3 and Robo4, respectively. (B) Semiquantitative RT-PCR and Western blotting analysis of ROBO1 and ROBO2 after transfection with siRNA for 24 hours in mouse calvaria osteoblasts. The SLIT3-stimulated (1.0 μg/ml) directional migration of mouse calvaria osteoblasts with or without Robo1 siRNA or Robo2 siRNA was assessed using a Boyden chamber system. SLIT3 treatment was for 24 hours, after which the invaded cell numbers were counted. (C) Analysis of Robo mRNA levels in mouse BMMs using semiquantitative RT-PCR. (D) Semiquantitative RT-PCR and Western blotting of ROBO1 and ROBO3 after transfection with siRNA for 24 hours in mouse BMMs. The SLIT3-mediated (1.0 μg/ml) suppression of osteoclastogenesis with or without Robo1 siRNA or Robo3 siRNA was assessed. SLIT3 treatment was for 4 days with 15 ng/ml M-CSF and 15 ng/ml RANKL, and TRAP-positive cells with more than 3 nuclei were counted. (E) Histomorphometric analyses of the femurs of 25-week-old male Robo1^–/– mice and WT littermates (n = 4–5 per group). Data are presented as mean ± SEM. In vitro experiments were performed 3–4 times independently. *P < 0.05 vs. untreated control or WT mice using the Mann-Whitney U test or Kruskal-Wallis test followed by Bonferroni’s correction.

Figure 5. Osteoclast-derived SLIT3 is indispensable for normal bone mass in vivo.

(A) Micro-CT analyses of the femurs of 16-week-old male Slit3_nestin^–/– mice and their Slit3^fl/fl littermates (n = 4–5 per group). (B) Micro-CT analyses of the femurs of 16-week-old male Slit3_col2.3^–/– mice and their Slit3^fl/fl littermates (n = 4–6 per group). (C) Micro-CT analyses of the femurs of 16-week-old male Slit3_ctsk^–/– mice and their Slit3^fl/fl littermates (n = 4–5 per group). (D) Von Kossa staining and histomorphometric analyses including calcein double-labeling of the femurs of 16-week-old male Slit3_ctsk^–/– mice and their Slit3^fl/fl littermates (n = 4–5 per group). Scale bars: 500 μm (left panels); 10 μm (right panels). Data are presented as mean ± SEM. *P < 0.05 vs. littermate control using the Mann-Whitney U test.

Figure 6. Effect of LRRD2 of human SLIT3 on bone mass in OVX mice.

(A) Directional migration of mouse calvaria osteoblasts upon treatment with the same molar concentration of SLIT3 (1.0 μg/ml = 10 nM) and LRRD2 for 24 hours using a Boyden chamber system. The invaded cell numbers were counted. (B) Proliferation of mouse calvaria osteoblasts in the presence of SLIT3 or LRRD2 for 48 hours assessed using a BrdU incorporation assay. (C) TRAP staining of mouse BMMs exposed to 15 ng/ml M-CSF and 15 ng/ml RANKL in the presence of SLIT3 or LRRD2 for 4 days. TRAP-positive cells with more than 3 nuclei were counted. (D) Von Kossa staining and histomorphometric analyses including calcein double-labeling of the femur of sham-operated, OVX, and LRRD2-treated OVX mice (n = 7 per group). The female C57BL/6J mice were OVX at 8 weeks of age, and 2 μg LRRD2 was injected via the tail vein twice a day (mean 0.192 mg/kg/day) from 12 weeks of age for 4 weeks. The same volume of saline was injected in the other groups. Mice were then sacrificed for analyses at 16 weeks of age. Scale bars: 500 μm (left panels); 10 μm (right panels). Data are presented as mean ± SEM. In vitro experiments were performed 3 times independently. *P < 0.05 vs. untreated control or between the indicated groups using the Mann-Whitney U test or Kruskal-Wallis test followed by Bonferroni’s correction. ^†P < 0.05 vs. 5 nM-treated group using Mann-Whitney U test.