Osteoclast-derived exosomes inhibit osteogenic differentiation through Wnt/β-catenin signaling pathway in simulated microgravity model
Graphical abstract
Introduction
During human spaceflight, astronauts are compelled to face several biological risks due to the hostile environment, such as microgravity, electromagnetic radiation and vacuum [[1], [2], [3]]. As the main stressful component, microgravity is well known to induce physiological changes in astronauts, including osteoporosis [[4], [5], [6]], skeletal muscle atrophy [7,8], immune system impairment [9,10] and other disorders. It is considered that osteoporosis under microgravity conditions is the most well recognized and prominent phenomenon [[11], [12], [13], [14]]. The force of gravity may maintain the balance of calcium metabolism and the mechanical environment of bone [15]. Thus, microgravity during spaceflight could disrupt bone homeostasis in the skeleton of astronauts and lead to bone weakness with a higher risk of osteoporosis and potential fracture for astronauts on their return to Earth [16]. Identification of the effect of microgravity on the bone is a considerable health challenge for astronauts. Consequently, to prevent microgravity-related osteoporosis, the effects of exercise and supplementation, the efficacy of hormone and antiresorptive drugs on bone metabolism have been widely studied over the past decade [[17], [18], [19]]. However, the mechanism of osteoporosis in microgravity has not been entirely elucidated. It requires understanding the cellular, molecular and systemic mechanisms as well as the underlying pathways involved in bone remodeling. The pathological mechanism may result from the imbalance of bone remodeling, which is a highly coordinated process involving bone formation and resorption [20,21].
Bone remodeling is a well-known dynamic cycle that involves activation, resorption, reversal and formation phases [22]. The balance of bone remodeling is maintained based on communication between several original cells, such as osteoclasts, osteoblasts, osteocytes and mesenchymal stem cells [23,24]. Among these types of cells, osteoclasts and osteoblasts, derived from multinucleated cells and mesenchymal stem cells, respectively, are responsible for bone resorption and formation [[25], [26], [27], [28], [29]]. The interaction between osteoclast-osteoblast occurs in a basic multicellular unit (BMU), which is the transient functional grouping of cells in bone [30]. It is recognized that osteoclast-osteoblast communication has three modes [31,32]. The first mode is direct contact, which allows membrane-bound ligands and receptors to interact and initiate intracellular signaling. Second, there is a gap junction between the two cell types, allowing small water-soluble molecules to cross over. It can also occur through diffusible paracrine factors containing cytokines, growth factors, and chemokines [33].
Recent studies have demonstrated that the critical factors involved in bone remodeling are packaged in exosomes, a type of extracellular vesicle [34,35]. Exosomes, which are membrane-bound vesicles produced by almost all cell types, are currently receiving increasing attention [36,37]. Consecutive research has considered exosomes and their messenger activity as an innovative mechanism of intercellular communication in various physiological and pathological processes, such as bone remodeling, vascular remodeling, tumor metastasis, and neuron signal transduction [[38], [39], [40], [41]]. Recent studies have shown that exosomes (average diameter 30 nm-100 nm) can also be secreted by bone-related cells, including osteoclasts, osteoblasts and osteocytes [42]. These bone cell-derived exosomes have been demonstrated to serve as mediators of intercellular communication in the bone microenvironment. Bone cell-derived exosomes can transfer their functional components such as proteins, microRNAs, and enzymes to adjacent or distant bone-related cells. Accumulating evidence indicates the involvement of exosomes in regulating bone remodeling mainly via transfer of the critical molecules required for communication between osteoclasts and osteoblasts [[43], [44], [45]]. Given this, exosomes may represent a novel approach for osteoclast and osteoblast communication, by which they coordinate their functions in specific physiological environments by delivering genetic information.
Evidence from in vivo and in vitro studies showed that both osteoclasts and osteoblasts could perceive and respond to the alteration of gravity [[46], [47], [48], [49]]. Moreover, the number of osteoblasts is decreased concurrently with enhanced osteoclastic activity in the space environment [50]. However, challenges still exist regarding whether osteoclasts can release exosomes for intercellular communication and regulate bone remodeling in microgravity environments. Our previous study demonstrated that osteoclast precursor cells have a direct adverse effect on osteoblasts under simulated microgravity conditions. Subjected to simulated microgravity conditions, conditioned medium from osteoclast precursor cells inhibited the activity of osteoblasts [51]. Recently, primary rat and cultured mouse osteoclast cell line were described as releasing exosomes [34]. Therefore, we hypothesized that exosomes derived from osteoclasts might represent a novel strategy for osteoblast-osteoclast communication under microgravity conditions. In this study, we investigated the role of exosomes from RAW264.7 cell-derived osteoclasts in the regulation of osteoblastic differentiation of MC3T3-E1 cells and its bone formation activity using a random positioning machine (RPM), which is a three-dimensional clinostat.
In this study, exosomes isolated from cultures of RAW264.7 cell-derived osteoclasts in RPM play a critical role in the regulation of osteoblastic proliferation, cell cycle, and differentiation through downregulating osteoblast-specific gene expression related to Wnt/β-catenin signaling. These results not only demonstrated the role of exosomes in communication between osteoblasts and osteoclasts in simulated microgravity but also suggested a potential pathway for diagnosis and therapy for osteoporosis of astronauts in space flight.
Section snippets
Cell culture and the random positioning machine (RPM) system
The murine osteoclast precursor cell line RAW264.7 [52] and the osteoblast-like cell line MC3T3-E1 [53] were purchased from the Cell Collection Center of Shanghai, which obtained the cell lines initially from American Type Culture Collection (Manassas, VA, USA). Both RAW264.7 and MC3T3-E1 cells were maintained in α-MEM (Gibco, Carlsbad, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, USA), 2 mmol/L l-glutamine, 2.2 g/L sodium bicarbonate, 100 units/ml penicillin
RPM system elevated osteoclastogenesis of RAW 264.7 cells
The osteoclast precursor cell line RAW264.7 cells were seeded in culture plates containing RANKL (50 ng/ml) in RPM for their differentiation until reaching maturity (Fig. 1A). As shown in Fig. 1B, a greater number of giant TRAP-positive multinucleated cells were observed in the RPM system culture than in the normal gravity control cell cultures, which had fewer and smaller multinucleated cells. The number and corresponding area of TRAP-positive multinucleated cells were quantified, as shown in
Discussion
Osteoporosis is the first proven pathological problem in astronauts returning from space missions including Gemini, Apollo, and Skylab. It is a well-known condition with alterations in bone microarchitecture and decreases in bone mass. Evidence indicates that astronauts invariably suffer a severe bone loss during space missions because of disruptions in the dynamic balance of bone remodeling in spaceflight or an altered gravitational environment [5,6]. The increased osteoclastogenesis observed
Declaration of interest statement
The authors report no conflicts of interest.
Acknowledgments
We gratefully acknowledge financial support from the National Natural Science Foundation of China (31500688, 81502465 and 51477141), the Fundamental Research Funds for the Central Universities (3102016OQD042), the Opening Foundation of the State Key Laboratory of Space Medicine Fundamentals and Application, Chinese Astronaut Research and Training Center (SMFA12K05), the Top International University Visiting Program for Outstanding Young Scholars of Northwestern Polytechnical University and the
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