Identification of a Novel l-Serine Analog That Suppresses Osteoclastogenesis in Vitro and Bone Turnover in Vivo

Takuya Ogawa,Tatsuo Takeya,Keiichiro Yogo,Norihiro Ishida-Kitagawa,Anton Bahtiar,Takahiro Matsumoto,Motofusa Akiyama,Takashi Nakamura

doi:10.1074/jbc.m109.058933

Takuya Ogawa, Tatsuo Takeya + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.058933

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Abstract

Osteoclasts are multinucleated giant cells with bone resorbing activity. We previously reported that the expression of the transcription factor NFAT2 (NFATc1) induced by receptor activator of NF-kappaB ligand (RANKL) is essential for the formation of multinucleated cells. We subsequently identified L-Ser in the differentiation medium as necessary for the expression of NFAT2. Here we searched for serine analogs that antagonize the function of L-Ser and suppress the formation of osteoclasts in bone marrow as well as RAW264 cells. An analog thus identified, H-Ser(tBu)-OMe x HCl, appeared to suppress the production of 3-ketodihydrosphingosine by serine palmitoyltransferase, and the expression and localization of RANK, a cognate receptor of RANKL, in membrane lipid rafts was down-regulated in the analog-treated cells. The addition of lactosylceramide, however, rescued the osteoclastic formation. When administered in vivo, the analog significantly increased bone density in mice and prevented high bone turnover induced by treatment with soluble RANKL. These results demonstrate a close connection between the metabolism of L-Ser and bone remodeling and also the potential of the analog as a novel therapeutic tool for bone destruction.

Highlights

The differentiation medium contained seven nonessential amino acids (L-Ala, L-Asn, L-Asp, L-Glu, L-Gly, L-Pro, and L-Ser), no other amino acid showed such a characteristic property
We identified a novel serine analog, H-Ser(tBu)-OMe1⁄7HCl, that suppressed osteoclastogenesis in vitro by down-regulating RANK expression as well as its localization in membrane lipid rafts and the subsequent RANKL/RANK signaling cascade
We examined the effect of the analog dosage on bone turnover in terms of bone density using RANKL-treated as well as control (GST-treated) C57BL6 mice

Summary

Introduction

The differentiation medium contained seven nonessential amino acids (L-Ala, L-Asn, L-Asp, L-Glu, L-Gly, L-Pro, and L-Ser), no other amino acid showed such a characteristic property. Bone marrow cells were prepared and induced to form MN cells in the presence of each substance at the indicated concentration, as described under “Experimental Procedures.” The morphology of cells and numbers of TRAP-positive MN cells are shown.

Results

Conclusion