GM-CSF Regulates Fusion of Mononuclear Osteoclasts into Bone-Resorbing Osteoclasts by Activating the Ras/ERK Pathway

Abstract

Osteoclasts are multinucleated cells that are formed by the fusion of mononuclear osteoclasts, which is an essential process in bone resorption leading to bone remodeling. Herein we show that GM-CSF promoted the fusion of prefusion osteoclasts (pOCs). The expression of GM-CSF receptor-α was significantly up-regulated at the fusion stage of pOCs induced by RANKL. GM-CSF induced the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which was mediated by inducing NFATc1 via induction of c-Fos. The expression of c-Fos and NFATc1 was regulated by the ERK signaling pathway. Inhibition of ERK and NFATc1 suppressed the expression of DC-STAMP and led to the fusion inhibition of pOC. However, retrovirus-mediated expression of NFATc1 in pOCs rescued the defect in pOC fusion, despite the presence of U0126 and cyclosporin A. GM-CSF-stimulated pOCs had an intact actin ring and could resorb bone. Importantly, pOCs infected with constitutively active MEK adenovirus expressed c-Fos and NFATc1, followed by the binding of NFATc1 to the DC-STAMP promoter, which enables its transcription and expression. Constitutively active MEK-infected pOCs are able to resorb bone by undergoing cell-cell fusion. Taken together, our results demonstrated that GM-CSF induced fusion of pOCs to form multinucleated osteoclasts, making the osteoclast capable of bone resorption.

Effect of interferon-γ on the fusion of mononuclear osteoclasts into bone-resorbing osteoclasts

“…DC-STAMP is a seven-transmembrane protein that was originally identified in dendritic cells (26). It has been reported that GM-CSF significantly induces pOC fusion by inducing DC-STAMP expression (21). This finding is consistent with ours, in which only DC-STAMP expression is significantly increased in the pOCs treated with IFN-γ; however, a number of studies have reported the role of fusion-related molecules in the multinucleation of pOCs (16)(17)(18)(19).…”

“…Bone marrow cells (BMCs) were collected from 5-week-old male ICR mice as described previously (21). The BMCs were suspended in α-MEM containing 10% FBS and antibiotics, and were cultured with M-CSF (10 ng/ml) for 1 day.…”

“…Osteoclast differentiation is exquisitely regulated by various cytokines, including RANKL, interleukins, interferons (IFNs), TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (6). We have reported recently that although GM-CSF greatly inhibits osteoclast differentiation, it can induce the fusion of pOCs into bone-resorbing osteoclasts (21). These findings indicated that although negative regulators of osteoclast differentiation can inhibit commitment into TRAP-positive cells, they might positively regulate other processes, such as cell-cell fusion and bone resorption.…”

The P2X7 Receptor and Pannexin-1 Are Both Required for the Promotion of Multinucleated Macrophages by the Inflammatory Cytokine GM-CSF

“…Notably, multinucleated MA have been observed in tuberculosis (5), yeast infection (6), HIV infection (7), failure of orthopedic implants (8), and sarcoidosis (5,9). Accordingly, a number of cytokines produced by immune cells including GM-CSF, M-CSF, IL-4, IL-13, and IL-6, alone or in combination, have been reported to stimulate the appearance of multinucleated MA in various cell culture systems (4,(10)(11)(12)(13)(14). Despite the widespread observation of MA fusion in vitro and in vivo, the mechanisms underlying this process remain poorly understood.…”

IL-4-mediated transcriptional regulation of human CYP2E1 by two independent signaling pathways

“…A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 shown that NFATc1 expression was inhibited by the MEK inhibitor U0126 [32]. We could partially inhibit the IL-4 induction of CYP2E1 with the same MEK inhibitor (U0126), indicating the involvement of this protein kinase (Supplemental data, Fig.…”