Abstract
Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3’s involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that STAT3 plays a pivotal role in RANKL-induced osteoclast formation, and that MSM can attenuate RANKL-induced osteoclastogenesis by blocking both NF-kB and STAT3 activity.
Highlights
Bone remodeling describes the restructuring of existing bone, which is a delicately controlled balance between bone formation by osteoblasts and resorption by osteoclasts [1]
We investigated the effect of MSM on receptor activator NF-kB ligand (RANKL)-induced bone resorption during osteoclastogenesis in bone marrow derived macrophages (BMMs). 100 mM MSM suppressed RANKL-induced bone resorption by 75% (Fig 1C)
We recently found that MSM enhances osteoblast differentiation in MSCs through activation of STAT5b
Summary
Bone remodeling describes the restructuring of existing bone, which is a delicately controlled balance between bone formation by osteoblasts and resorption by osteoclasts [1]. RANKL-induced activation of RANK causes TNF receptor-associated factor 6 (TRAF6) recruitment in osteoclast precursor cells [4] and the sequential activation of mitogenactivated protein kinases (MAPKs) involving extracellular signaling-related kinase (ERK), p38, and Jun N-terminal kinase (JNK), and transcription factors such as nuclear factor-kappa B (NF-κB), activating protein 1 (AP-1), nuclear factor of activated T cells (NFATc1), and c-Fos [5]. The activation of these signaling effectors induces the expression of osteoclastic genes such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (Cts K), and matrix metalloproteinase 9 (MMP-9), whose activities result in the development of multinucleated bone-resorbing osteoclasts [5,6]. Recent data demonstrated a dual role for STAT3 depending on cell type (osteoblast or osteoclast) and its phosphorylation status [10]
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