Osteoclast-secreted SLIT3 coordinates bone resorption and formation
Beom-Jun Kim, … , Ghi Su Kim, Jung-Min Koh
Beom-Jun Kim, … , Ghi Su Kim, Jung-Min Koh
Published March 5, 2018
Citation Information: J Clin Invest. 2018;128(4):1429-1441. https://doi.org/10.1172/JCI91086.
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Research Article Bone biology

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.

Authors

Beom-Jun Kim, Young-Sun Lee, Sun-Young Lee, Wook-Young Baek, Young Jin Choi, Sung Ah Moon, Seung Hun Lee, Jung-Eun Kim, Eun-Ju Chang, Eun-Young Kim, Jin Yoon, Seung-Whan Kim, Sung Ho Ryu, Sun-Kyeong Lee, Joseph A. Lorenzo, Seong Hee Ahn, Hyeonmok Kim, Ki-Up Lee, Ghi Su Kim, Jung-Min Koh

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Figure 2

SLIT3 stimulates osteoblast migration and proliferation.

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SLIT3 stimulates osteoblast migration and proliferation. (A) Quantitativ...
(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.