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 1

Increased SLIT3 production during osteoclast differentiation.

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Increased SLIT3 production during osteoclast differentiation. (A) SLIT3 ...
(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.