Disulfiram Attenuates Osteoclast Differentiation <i>In Vitro</i>: A Potential Antiresorptive Agent

<div><p>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 <i>in vitro</i>. 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.</p></div

DSF suppresses RANKL-induced activation of NF-κB.

<p>(A) DSF suppressed RANKL-induced NF-B luciferase activity. RAW264.7 cells stably expressing a NF-B-driven luciferase reporter construct were pre-treated with varying doses of DSF for 1 hr followed by rRANKL stimulation. Luciferase activity was assessed after 8 hrs of RANKL stimulation (mean ± SD; *: P<0.05, **: P<0.01, ***: P<0.001 against RL⁺). (B) DSF delayed IκBα degradation. Total cell lysates were extracted from BMMs treated with rRANKL for 0, 5, 10, 20, 30 and 60 mins in the presence or absence of DSF (200 nM). Proteins were separated on 12.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and immunoblotted sequentially with antibodies to IκBα and p65. β-actin was used as internal loading control. (C and D) DSF attenuated the nuclear translocation of p65. BMMs were treated with RANKL for 1 hr in the presence or absence of DSF (200 nM). Cytoplasmic and nuclear fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents, separated on 12.5% SDS-PAGE gel, and then transferred onto nitrocellulose membrane. Membrane was immunoblotted for p65. -actin and TBP were used as loading controls for cytoplasmic and nuclear fractions respectively. Results shown represent one of three independent experiments.</p

DSF attenuates RANKL-induced MAPK signaling in BMMs.

<p>Total cell lysates were extracted from BMMs treated with rRANKL for 0, 5, 10, 20, 30 and 60 mins (A) or for 0, 24, 48 and 72 hrs (B) in the presence or absence of DSF (200nM or 100nM). Proteins were separated on 12.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and immunoblotted sequentially with antibodies to different components of the MAPK and SAPK signaling pathways (ERK, JNK, p38, Src and Akt). β-actin was used as internal loading control. Results shown represent one of three independent experiments.</p

Primer sequences and cycle numbers for semi-quantitative RT-PCR.

<p>Primer sequences and cycle numbers for semi-quantitative RT-PCR.</p

DSF inhibited osteoclastogenesis during the early stage of differentiation <i>in vitro</i>.

<p>(A) Time and dose-dependent inhibitory effect of DSF on OC formation. During a typical 5-day culture of osteoclastogenesis, DSF treatment on days 1–2 are defined as the early stage and treatment on days 3–4 as the late stage. Freshly isolated M-CSF-dependent BMMs cultured in the presence of rRANKL (100ng/ml) and various concentrations of DSF (12.5 nM, 25nM, 50 nM and 100 nM) for 4 days were fixed with 4% PFA followed by TRAP staining for the visualization of multinucleated OCs (scale bar = 500μm). (B) The number and average size in areas (mm²) of TRAP-positive multinucleated OCs (≥3 nuclei) at the different time points were quantified (mean ± SD; *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: not significant against vehicle). (C) Effect of DSF on OC-specific gene expression. Total RNA was isolated from BMM cells cultured in the presence or absence of rRANKL and various doses of DSF (25, 50 and 100nM) for 4 days. cDNA was synthesized using 1μg of total RNA and subjected to PCR amplification using specific primers for OC-specific genes, CtsK, TRAP, DC-STAMP, Atp6v0d2 and housekeeping gene β-actin. PCR products were separated and analysed on 1.5% agarose gels. (D) The relative levels of gene expression was calculated as ratios against β-actin (mean ± SD; *: p<0.05, **: p<0.01, ***: p<0.001, ns: not significant against RL⁺).</p

DSF inhibited RANKL-induced NFATc1 signaling.

<p>(A) DSF attenuated RANKL-induced NFATc1 protein expression. Total cell lysates were extracted from BMMs treated with rRANKL for 0, 24, 48 and 72 hrs in the presence or absence of DSF (100nM). Proteins were separated on 10% SDS-PAGE gel, transferred onto nitrocellulose membranes, and immunoblotted with specific antibody to NFATc1. β-actin was used as internal loading control. (B) DSF dose-dependently inhibited RANKL-induced NFATc1 luciferase activity. RAW264.7 cells stably expressing a NFATc1-driven luciferase reporter construct was pre-treated with varying doses of DSF (25, 50, 100nM) for 1 hr followed by rRANKL stimulation. Luciferase activity was assessed after 24 hrs of RANKL stimulation (mean ± SD; *P<0.05, **P<0.01, ***P<0.001 against RL⁺). (C) DSF impaired NFATc1 downstream effector proteins CtsK and Atp6v0d2 expression. Total cell lysates were extracted from BMM-derived OCs cultured in the presence or absence of DSF (100nM). Proteins were separated on 12.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and immunoblotted with specific antibodies to CtsK and Atp6v0d2. -actin was used as internal loading control. Results shown represent one of three independent experiments.</p

V-ATPase proton pump is not the target of DSF.

<p>(A) DSF does not affect V-ATPase-mediated acidification in OCs. Image merge of green and red fluorescence spectra of acridine orange (AO) fluorescence quenching from BMM-derived OCs pre-treated for 12 hrs with various concentrations of DSF (6.25, 12.5, 25, 50, 100 and 200 nM), Baf (10 nM) or SaliPhe (100 nM) followed by incubation with 5 μg/ml AO for 15 mins at 37°C. Cells were first excited with wavelength of 485 nm. Fluorescence shift of AO from green to red indicate normal intracellular acidification. (B) Fluorescence intensity at excitation 485 nm and emission 535 nm was quantified using a spectrophotometer (mean ± SD; **: P<0.01, ****: P<0.0001 against vehicle). (C and D) DSF does not affect V-ATPase-mediated proton transport in isolated microsomes. Microsomes isolated from RAW264.7 cells was subjected to AO proton transport assay in the presence or absence of DSF (100 nM), Baf (10 nM) or SaliPhe (100 nM). Proton transport was initiated by the addition of ATP and influx was detected by the fluorescence intensity measured with excitation 485 nm and emission 535 nm and results are represented as the initial rate of acidification (-∆F/∆t as calculated from the slope generated by the first 60 seconds after ATP supplement and normalized to vehicle). Results shown represent one of three independent experiments.</p