Received for publication January 18, 2007.
Revised May 11, 2007.
Accepted for publication May 15, 2007.

Suppression of osteoclastogenesis by N,N-dimethyl-D-erythro-sphingosine: A SPHINGOSINE KINASE INHIBITION-INDEPENDENT ACTION

Abstract

N,N-dimethyl-D-erythro-sphingosine (DMS) competitively inhibits sphingosine kinase (SPHK) and has been widely used to assess the role of SPHK during cellular events including motility, proliferation, and differentiation. In the present study, the effect of DMS on the differentiation of bone marrow macrophages (BMMs) to osteoclasts was investigated. When the osteoclast precursor cells were treated with DMS, the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis was completely blocked. Surprisingly, however, knock-down of SPHK by siRNA in BMMs did not reduce osteoclastogenesis. Furthermore, both overexpression of SPHK and exogenous addition of sphingosine-1-phosphate, the product of SPHK activity, failed to overcome the anti-osteoclastogenic effect of DMS. These results suggest that DMS inhibited osteoclastogenesis independently of SPHK. Subsequent characterization of the DMS-mediated suppression of osteoclastogenesis revealed that DMS did not affect RANKL-induced activation of JNK, p38, NF-B, and Ca²⁺ oscillation. On the other hand, DMS strongly inhibited two separate signaling pathways, the RANKL-induced activation of ERK and Akt, which eventually converged on the transcription factors c-Fos and NFATc1. There was significant increase in the osteoclast formation in the presence of DMS when BMMs were overexpressed with c-Fos, suggesting that c-Fos was a critical downstream target of DMS for the inhibition of osteoclastogenesis. Taken together, our data demonstrate that DMS has an anti-osteoclastogenic function independently of its SPHK inhibitory activity. Considering previously reported anti-cancer properties of DMS, our study may also propose that DMS is an ideal drug candidate for bone metastases, for which osteoclastic bone-resorption is crucial.

Key words: MAP Kinase, AP-1, NFAT, NFkappaB