Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease
Adel Ersek, … , Nicole J. Horwood, Anastasios Karadimitris
Adel Ersek, … , Nicole J. Horwood, Anastasios Karadimitris
Published April 27, 2015
Citation Information: J Clin Invest. 2015;125(6):2279-2292. https://doi.org/10.1172/JCI59987.
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Research Article Oncology

Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease

Abstract

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell–derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Authors

Adel Ersek, Ke Xu, Aristotelis Antonopoulos, Terry D. Butters, Ana Espirito Santo, Youridies Vattakuzhi, Lynn M. Williams, Katerina Goudevenou, Lynett Danks, Andrew Freidin, Emmanouil Spanoudakis, Simon Parry, Maria Papaioannou, Evdoxia Hatjiharissi, Aristeidis Chaidos, Dominic S. Alonzi, Gabriele Twigg, Ming Hu, Raymond A. Dwek, Stuart M. Haslam, Irene Roberts, Anne Dell, Amin Rahemtulla, Nicole J. Horwood, Anastasios Karadimitris

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Figure 1

GSL inhibitors inhibit osteoclastogenesis and bone resorption in vitro.

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GSL inhibitors inhibit osteoclastogenesis and bone resorption in vitro. ...
(A) Mouse BM cells were cultured in the presence of 25 ng/ml M-CSF with 50 ng/ml RANKL (Control) and with or without d-PDMP (5, 20, or 50 µM), NB-DNJ, NB-DGJ, or N-OD-DNJ (5, 50, or 500 µM) in 48-well plates for 5 days, and TRAP-positive OCs were counted and shown as cumulative results from 3 different experiments. (B) Cultures with iminosugar inhibitors at 500 µM and d-PDMP at 20 µM were photographed (original magnification, ×40). (C and D) Mouse BM cells were cultured with 25 ng/ml M-CSF and 50 ng/ml RANKL in 48-well plates. On day 3 d-PDMP (20 µM) or NB-DNJ, NB-DGJ, or N-OD-DNJ (each at 500 µM) was added. Cells were cultured for another 48 hours before being fixed and stained for TRAP and counted (C) and phalloidin stained to identify mature OCs by the formation of actin rings (D) (original magnification, ×40). (E and F) Mouse BM cells cultured with M-CSF and RANKL on dentine slices with 5 µM d-PDMP or 50 µM iminosugars added on day 1 were evaluated on day 14 when resorption lacunae on dentine slices were visualized with methylene blue (original magnification, ×4). (F) Resorption area is expressed as a percentage of the dentine surface area. A representative experiment of three, each performed in triplicate assays, is shown for C and F. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001 versus control).