Original Full Length ArticleAfamin secreted from nonresorbing osteoclasts acts as a chemokine for preosteoblasts via the Akt-signaling pathway
Highlights
► Osteoclasts themselves, not their resorptive activities or precursors, play a central role in osteoclast–osteoblast communication during bone remodeling. ► Osteoclasts in the early differentiation stage secrete potential coupling factors that attract osteoblastic lineages. ► Afamin secreted from differentiated osteoclasts is a possible coupling factor for preosteoblast migration. ► Chemotactic effects of afamin are mediated by Akt-signaling pathway.
Introduction
Bone is a dynamic tissue that is constantly remodeling and can undergo regeneration throughout life in response to biochemical and mechanical signals. This continuous remodeling occurs through a dynamic process of osteoclastic breakdown and osteoblastic rebuilding. The balance between bone resorption and formation is normally tightly controlled in a local, coordinated, and sequential manner, which is referred to as the “coupling phenomenon” [1]. Imbalance between these two processes leads to metabolic bone disorders, such as osteoporosis and osteopetrosis [2], [3].
The mechanisms by which osteoblasts induce osteoclastic differentiation are well elucidated. Osteoblasts express macrophage colony-stimulating factor (M-CSF), which is required for the proliferation and survival of osteoclastic precursors, and receptor activator of NF-κB ligand (RANKL) to trigger differentiation and control osteoclastogenesis [4]. On the other hand, how osteoblasts are recruited to transition-phase sites and how bone-forming processes are controlled within the basic multicellular unit (BMU) are relatively less understood. Traditionally, osteoclastic activity as a coupling factor has been the focus of research on bone resorption. Osteoclastic bone resorption may liberate factors embedded in the bone matrix, such as transforming growth factor (TGF)-β, bone morphogenetic proteins (BMPs), and insulin-like growth factor (IGF)-II [5], [6], which in turn activate osteoblastic bone formation. In addition, a recent report showed that bidirectional interactions between ephrin-B2, which is expressed by osteoclasts, and its Eph-B4 receptor, which is expressed by osteoblasts, may facilitate the transition from bone resorption to bone formation, suggesting that cell-surface factors on osteoclasts function as potential coupling factors [7], [8].
Interestingly, several lines of evidence suggest that osteoclast-derived factors are indispensable for coupling. Based on osteopetrosis models, Karsdal et al. [9] hypothesized that the osteoclasts themselves can induce bone formation independent of bone matrix signals. Patients with osteopetrosis caused by impaired acidification of the resorption lacunae, as seen when mutations occur in osteoclast a3-V-ATPase or chloride channel ClC-7, have unaltered or even increased bone formation despite reduced bone resorption [10]. This hypothesis was demonstrated in an in vitro study that showed that conditioned media (CM), which was collected from nonresorbing osteoclasts, dose-dependently induces osteoblastic differentiation [11]. In addition, a recent study revealed that the supernatant collected from human nonresorbing-osteoclast cultures can induce migration and osteogenic differentiation in human mesenchymal stem cells [12].
The other question that needs to be answered to understand the coupling phenomenon is which stages of bone formation are affected by the coupling factors. The answer could be presumed from the observations in clinical studies that compared bone histomorphometry before and after bisphosphonate treatment. Recker et al. [13] reported that zoledronate suppresses the volume-referent bone formation rate (BFR) via the coupling phenomenon. However, the mineral apposition rate (MAR), which reflects the bone-forming capacity of individual teams of osteoblasts at the BMU level, was not reduced by treatment. These interesting results are consistent for all types of bisphosphonate [14], [15], [16], implying that coupling factors mainly affect the stages related to the osteoblastic number on the bone-forming surface rather than osteoblastic bone-forming activities.
In the present study, we established an in vitro system for the coupling phenomenon and investigated when the coupling factors are produced during osteoclastic bone resorption, and how they affect the osteoblastic number. Furthermore, by using a fractionated secretomics method, we identified afamin as one of novel osteoclast-derived coupling factors.
Section snippets
Materials and reagents
Antibodies against Akt, Rac, Cdc42, and RhoA were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NFATc1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against afamin and recombinant afamin were obtained from Professor Hans Dieplinger at Innsbruck Medical University, Austria [17], and detailed information was described in the Supplementary material and methods. Inhibitors of Rho kinase (Rock; Y27632) and Akt (LY294002) were
Osteoclasts in the early differentiation stage secrete potential coupling factors that attract osteoblast precursors
In order to establish an in vitro system for investigating the coupling phenomenon, variable culture conditions for osteoclastogenesis and bone resorption were generated. We treated BMMs with 100 ng/mL RANKL for 2 days and 6 days in the presence of 30 ng/mL M-CSF to induce early- and late-osteoclast differentiation, respectively. To induce the resorption stage, BMMs were incubated with 100 ng/mL RANKL and mouse femurs for 10 days in the presence of 30 ng/mL M-CSF. Osteoclast differentiation and bone
Discussion
In an effort to identify novel osteoclast-derived anabolic factors, independently of the release of bone matrix signals, we established an in vitro system for investigating the coupling phenomenon. We observed that the supernatant collected from early-differentiated osteoclasts predominantly stimulated preosteoblast mobilization. By further performing proteomic analysis using LC-MS/MS, we identified afamin as one of osteoclast-derived chemokines that affect preosteoblasts, and suggested that
Acknowledgments
This study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (project no.: A110536) and a grant from the Asan Institute for Life Sciences, Seoul, Korea (project no.: 2011‐523).
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Beom-Jun Kim and Young-Sun Lee contributed equally to this work.