Abstract
Disulfiram (DSF), a cysteine modifying compound, has long been clinically employed for the treatment of alcohol addiction. Mechanistically, DSF acts as a modulator of MAPK and NF-κB pathways signaling pathways. While these pathways are crucial for osteoclast (OC) differentiation, the potential influence of DSF on OC formation and function has not been directly assessed. Here, we explore the pharmacological effects of DSF on OC differentiation, activity and the modulation of osteoclastogenic signaling cascades. We first analyzed cytotoxicity of DSF on bone marrow monocytes isolated from C57BL/6J mice. Upon the establishment of optimal dosage, we conducted osteoclastogenesis and bone resorption assays in the presence or absence of DSF treatment. Luciferase assays in RAW264.7 cells were used to examine the effects of DSF on major transcription factors activation. Western blot, reverse transcription polymerase chain reaction, intracellular acidification and proton influx assays were employed to further dissect the underlying mechanism. DSF treatment dose-dependently inhibited both mouse and human osteoclastogenesis, especially at early stages of differentiation. This inhibition correlated with a decrease in the expression of key osteoclastic marker genes including CtsK, TRAP, DC-STAMP and Atp6v0d2 as well as a reduction in bone resorption in vitro. Suppression of OC differentiation was found to be due, at least in part, to the blockade of several key receptor activators of nuclear factor kappa-B ligand (RANKL)-signaling pathways including ERK, NF-κB and NFATc1. On the other hand, DSF failed to suppress intracellular acidification and proton influx in mouse and human osteoclasts using acridine orange quenching and microsome-based proton transport assays. Our findings indicate that DSF attenuates OC differentiation via the collective suppression of several key RANKL-mediated signaling cascades, thus making it an attractive agent for the treatment of OC-mediated disorders.
Highlights
Bone is a dynamic organ undergoing constant remodeling to ensure correct mineral homeostasis and to maintain proper mechanical strength [1]
And 2B, DSF dose-dependently reduced both the number and size/area of TRAP-positive OCs. This reduction in OC formation was primarily observed when DSF was administered to cultures during the early stage (Day 1–2), but not late stage (Day 3–4) of the differentiation process (Fig 2B). This indicates that the inhibitory action of DSF predominates during the early stage of receptor activators of nuclear factor kappa-B ligand (RANKL)-driven OC differentiation
To establish a mechanistic basis for the observed decrease in OC differentiation during the early stage of osteoclastogenesis, we examined the effect of DSF on key RANKL-mediated signaling cascades including mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) phosphorylation
Summary
Bone is a dynamic organ undergoing constant remodeling to ensure correct mineral homeostasis and to maintain proper mechanical strength [1]. Upon binding to its cognate receptor RANK (expressed on the surface of OC progenitors), RANKL triggers the activation of several downstream nuclear transcription factors among which nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein 1 (AP-1) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) are indispensable for OC differentiation [7]. During their RANKL-driven maturation, the OC plasma membrane is furnished with specialized molecular machinery such as vacuolar H+-type ATPases (V-ATPases) proton pumps and chloride channels which facilitates its unique bone-resorbing function. Certain V-ATPase subunits have been shown to relate to OC formation [11, 12]
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