The myokine irisin facilitates muscle-bone crosstalk and skeletal remodeling in part by its action on osteoblasts and osteocytes. In the current study we investigated whether irisin also directly regulates osteoclasts. In vitro, irisin (2-10 ng/mL) increased osteoclast differentiation in C57BL/6J bone marrow progenitors; this increase was blocked by a neutralizing antibody to integrin αVβ5. Irisin also increased resorption on several substrates in situ.RNAseq revealed differential gene expression induced by irisin including upregulation of markers for osteoclast differentiation and resorption, as well as osteoblast-stimulating 'clastokines'. In vivo, forced expression of the irisin precursor Fndc5 in murine muscle resulted in low bone mass and increased number of osteoclasts. Taken together, our work demonstrates that irisin acts directly on cultured osteoclast progenitors to increase differentiation and promote bone resorption. These actions support the tenet that irisin not only stimulates bone remodeling but may also be an important counter-regulatory hormone during exercise.
Introduction.Irisin is a peptide generated by proteolytic cleavage of fibronectin type III domain-containing 1 protein 5 (FNDC5), a membrane-bound protein highly expressed in skeletal muscle. Fndc5 expression increases in 2 response to acute bouts of exercise under regulation by PGC-1α, leading to a burst of circulating irisin 1 . Initially irisin 3 was described as a circulating hormone that induces thermogenesis in adipose tissue 2 , but more recent work has shown 4 a potent ability to modulate bone turnover. These effects support the tenet that irisin may be a key mediator of muscle-5 bone crosstalk during exercise. Initial studies demonstrated that irisin enhanced cortical bone formation and prevented 6 unloading-induced bone loss in vivo, and stimulated osteoblasts in vitro 3-5 . Conversely, genetic deletion of Fndc5 was 7 separately shown to block resorption-driven bone loss and maintain osteocyte function following ovariectomy; irisin 8 treatment in vitro also prevented osteocyte apoptosis and stimulated sclerostin and RANKL release, key promoters of 9 osteoclastogenesis, through the αVβ5 integrin receptor 6 . The present study addresses the hypothesis that irisin also 10 directly stimulates osteoclast differentiation and function in vitro and in vivo.
12Results and Discussion. First, we used continuous treatment with increasing doses of irisin (0, 2, 5,10 13 ng/mL) for 7-days in an in vitro osteoclast differentiation assay using primary marrow hematopoietic progenitors.14 These doses were based on previous work establishing the physiologic range of circulating irisin during and after 15 exercise 1 . We found a qualitative enhancement of both osteoclast number and size (Figure 1a), and significant 16 increases in osteoclast number across this dose range (2 ng/mL, 5 ng/mL, 10 ng/mL: P < .0001, 20 ng/mL: P = .044) 17 ( Figure 1b). Based on these results, we selected 10 ng/mL irisin for further experiments using both continuous (7 day...