Abstract
Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued.
Highlights
To further verify the role of the P38 and extracellular signal-regulated kinase (ERK) signaling pathways in irisin-induced osteogenic effects, osteoblasts were pretreated with phosphate-buffered saline (PBS), or the p38 inhibitor SB203580 (SB), or the ERK inhibitor U0126 (U0) for 30 mins, cell was cultured in the presence of r-irisin, and P38 and ERK phosphorylation were determined
We report that irisin, a novel identified exercise-induced myokine, could directly targets osteoblasts and promotes proliferation, differentiation, and mineralization though P38/ERK MAPK signaling pathways
Our study confirmed that both conditioned media (CM)-irisin and r-irisin can directly target osteoblast and enhance osteoblast proliferation
Summary
Preparation and Identification of irisin from cultured media. In order to study the role of irisin close to natural state, we used the method described previously[11]. The Alizarin Red staining intensity significantly enhanced after treatment with CM-irisin or r-irisin (Fig. 4B,F), and the amount of calcium deposition is increased in irisin group than control (Fig. 4D,H)) These results indicate that irisin promotes osteoblast differentiation and mineralization in vitro. To further verify the role of the P38 and ERK signaling pathways in irisin-induced osteogenic effects, osteoblasts were pretreated with PBS (control), or the p38 inhibitor SB203580 (SB), or the ERK inhibitor U0126 (U0) for 30 mins, cell was cultured in the presence of r-irisin, and P38 and ERK phosphorylation were determined. Representative images of MC3T3-E1 osteoblast treated with r-irisin (100 ng/ml) or PBS as control by alkaline phosphatase staining (E) and Alizarin Red staining (F) after culturing the cells in osteogenic medium for 14 days, the ALP activity (G) and Quantification of Alizarin Red S stain (H) was measured and normalized to the total protein content. Our results demonstrated that irisin promotes osteoblast proliferation and differentiation via the P38 and ERK signaling pathways
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