The bone microenvironment (BME) is the main hub of all skeletal related pathological events in osteosarcoma leading to tumor induced bone destruction, and decreasing overall bone quality and bone strength. The role of extra-cellular membrane vesicles (EMVs) as mediators of intercellular communication in modulating osteosarcoma-BME is unknown, and needs to be investigated. It is our hypothesis that osteosarcoma-EMVs contain pro-osteoclastogenic cargo which increases osteoclastic activity, and dysregulated bone remodeling in the osteosarcoma-BME. In this study, EMVs were isolated from the conditioned media of 143B and HOS human osteosarcoma cell cultures using differential ultracentrifugation. Nano-particle tracking analysis determined EMVs in the size range of 50-200 nm in diameter. The EMV yield from 143B cells was relatively higher compared to HOS cells. Transmission electron microscopy confirmed the ultrastructure of 143B-EMVs and detected multivesicular bodies. Biochemical characterization of 143B-EMVs detected the expression of bioactive pro-osteoclastic cargo including matrix metalloproteinases-1 and -13 (MMP-1, -13), transforming growth factor-β (TGF-β), CD-9, and receptor activator of nuclear factor kappa-β ligand (RANKL). Detection of a protein signature that is uniquely pro-osteoclastic in 143B-EMVs is a novel finding, and is significant as EMVs represent an interesting mechanism for potentially mediating bone destruction in the osteosarcoma-BME. This study further demonstrates that 143B cells actively mobilize calcium in the presence of ionomycin, and forskolin, and induce cytoskeleton rearrangements leading to vesicular biogenesis. In conclusion, this study demonstrates that 143B osteosarcoma cells generate EMVs mainly by mechanisms involving increased intracellular calcium or cAMP levels, and contain pro-osteoclastic cargo.
There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice.
Several studies suggest that the decline of muscle function with age can be mitigated by dietary supplementation with beta‐hydroxy‐beta‐methylbutyrate (HMB) or beta‐alanine (BA). The purposes of this study were to investigate the effects of: 1) HMB treatment on murine C2C12 myoblast and myotube cultures; and 2) dietary HMB, BA, and a combination thereof, on muscle contractility in late middle‐aged mice. In C2C12 myotubes, free acid HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of three separate SR agonists (P<0.05, ANOVA). HMB also improved C2C12 myoblast viability (P<0.05) and proliferation (P=0.017). Furthermore, an ex vivo muscle contractility study was performed on 48 C57BL/6nTac male mice (19 months old at end of study). For 8 weeks, mice were fed control AIN‐93M diet containing calcium HMB monohydrate, BA, or the combination thereof. In EDL muscle, the combination of HMB and BA showed significant increases in relative force (88.6 ± 2.2 mN vs. 79.8 ± 2.4 mN, P=0.025) and specific force (12.2 ± 0.4 N/cm2 vs. 10.8 ± 0.4 N/cm2, P=0.021) at submaximal frequency of stimulation. At maximum frequency of stimulation, BA showed a significant force increase over the control diet (202.0 ± 3.4 mN vs. 188.2 ± 4.6 mN, P=0.023). Effects in soleus muscle did not achieve statistical significance. Our findings suggest calcium signaling as a key pathway contributing to beneficial effects of HMB and BA on skeletal muscle.
The intricacy of cell interactions with their microenvironment and their ability to communicate at the autocrine, paracrine, and endocrine levels continues to be revealed. Until recently, the musculoskeletal system was believed to be directed primarily by a relationship of proximity and function, dictated in large part by mechanical forces and the work of gravity. The purpose of this review is to provide an overview of the biomechanical theory of bone‐muscle interaction and to present the substantial evolution in our understanding of bones and muscles as secretory organs; including new evidence from our research labs in support of this biochemical crosstalk between bone and muscle cells. We propose that the mechanical and biochemical modes of interaction between bones and muscles are complementary and interwoven. In fact, we hypothesize that one tissue action might prime the other tissue function, thereby contributing for optimal functional unit. This perspective provides a fertile new venue for bone‐muscle crosstalk exploration, as well as the development of innovative therapies targeting musculoskeletal diseases, which currently afflict nearly 2 billion people worldwide. Grant Funding Source: Supported by NIH P01 AG039355‐01‐A1
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