Biochemical and Biophysical Research Communications
The effects of myokines on osteoclasts and osteoblasts
Introduction
Muscle and bone are the largest tissues of non-obese humans and share the common purposes of protecting internal organs and enabling movement [1]. Interestingly, clinical observations indicate that changes in muscle and bone health are strongly correlated throughout an individual's lifetime [2,3]. For example, muscle atrophy and bone loss are often present in individuals of similar age groups [4,5]. Because both sarcopenia and osteoporosis are well-known risk factors for fragility fractures [6], a better understanding about muscle–bone interactions underlying the parallel changes is essential for effective prevention of fractures.
Although interactions between muscle and bone can be bi-directional, their highly integrated nature has been conventionally explained by muscle force-generated mechanical signals, which transduce anabolic activity in the adjacent bone [7,8]. Importantly, muscle has recently received much attention as an endocrine organ that regulates other biological targets, including the pancreas, liver, and adipose tissue [9]. Therefore, besides the mechanostat theory, there is a possibility that muscle-secreting factors—defined as “myokines”—biochemically affect bone metabolism in a paracrine manner [10,11]. When open tibial fractures were covered with muscle flap in a murine model, the rate and quality of bone repair were markedly improved compared with fractures covered with equally vascularized fasciocutaneous tissue [12,13], which supports the aforementioned hypothesis. However, despite this plausibility, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. With an aim of further understanding how muscle interacts with bone, we performed in vitro as well as animal experiments using conditioned media (CM) from C2C12 myoblast and myotube cultures.
Section snippets
Collection of C2C12 myoblast and myotube CM
Mouse C2C12 myoblasts were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% FBS, 20 mM HEPES, 2 mM l-glutamine, and antibiotics (Life Technologies Corp., Carlsbad, CA) at 37 °C in a humidified atmosphere comprising 5% CO2. CM was collected after further incubation for 24 h in serum- and phenol red-free media, and these were regarded as myoblast CM. To induce myogenic differentiation, cells were grown to 90% confluency in
Suppression of osteoclastogenesis and in vitro bone resorption using myotube CM
The effects of myotube CM on the biology of osteoclasts were compared with those of myoblast CM and non-CM (control). When primary mouse BMMs were cultured with various CM concentrations in the presence of M-CSF and RANKL, 10%, 25%, and 50% myotube CM significantly inhibited osteoclast differentiation, as shown by the results of TRAP staining (Fig. 1A). Because the effect on osteoclastogenesis was already at a maximum with 25% myotube CM, subsequent experiments on osteoclasts were performed
Discussion
Since Pederson et al. [18] had first coined the term “myokines” to describe cytokines or other peptides that are produced and secreted by muscle fibers, several lines of evidence have come to support the role of muscle as an endocrine organ [9,10]. In addition, in the present study, we have demonstrated the distinct effects of myotube versus myoblast CM on bone cells. Myotube CM inhibited in vitro bone resorption through the suppression of osteoclastogenesis and osteoclastic resorptive
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [grant numbers 2016M3A9E8941329 and 2019R1A2C2006527].
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Jin Young Lee and So Jeong Park contributed equally to this work.