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Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB

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Abstract

Receptor activator of NF-κB (RANK) is a member of the tumor necrosis factor receptor (TNFR) family expressed in osteoclast precursors, and RANK–RANK ligand (RANKL) signaling is a key system for differentiation, activation and survival of osteoclasts. Here, we report the identification of a novel alternative splicing variant of mouse RANK gene (vRANK) that contains a new intervening exon between exon 1 and exon 2 of mouse full-length RANK (fRANK) mRNA. Since this novel exon contains the stop codon, vRANK encodes truncated amino acids that have a portion of the signal peptide of fRANK and an additional 19 amino acids that show no homology to previously reported domains. By transient transfection studies with vRANK–GFP and –Flag expressing constructs, vRANK was found localized mostly in the cytoplasm and partly in the cell membrane, but was not secreted into the culture supernatant. Under the stimulation of various factors, the expression of vRANK mRNA was almost parallel to that of fRANK in RAW264.7 cells not treated with M-CSF. Overexpression of vRANK, on the other hand, decreased TRACP (a marker of osteoclasts) mRNA expression as well as the number of TRACP-positive multinucleated giant cells. While the mRNA expression levels of NFATc1 (a master transcriptional factor of the osteoclast differentiation program) were not affected, apoptotic cells increased significantly in vRAN K-transfected cells treated with sRANKL. Taken together, these results suggest that vRANK is a novel osteoclast suppressor that reduces the number of RANKL-induced mature osteoclasts mainly by negating the anti-apoptotic effect of RANKL.

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Abbreviations

RANK:

Receptor activator of NF-κB

RANKL:

Receptor activator of NF-κB (RANK) ligand

GFP:

Green fluorescent protein

M-CSF:

Macrophage colony-stimulating factor

TRACP:

Tartrate-resistant acid phosphatase

NFAT:

Nuclear factor of activated T cells

OPG:

Osteoprotegerin

TNF:

Tumor necrosis factor

TRAF:

Tumor necrosis factor receptor-associated factor

JNK:

c-Jun N-terminal kinase

ERK:

Extracellular signal-regulated kinase

Akt:

AKR mouse T-cell lymphoma

LPS:

Lipopolysaccharide

MITF:

Microphthalmia-associated transcription factor

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Acknowledgments

The authors thank Mr. Shuichi Matsuda and Ms. Noriko Sakamoto for excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (16590278, 19390100 and 19659086 to S.K., 20790283 to T.K., 18590372 and 21590419 to R.K., and 21790351 to K.M.).

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Authors and Affiliations

  1. Division of Diagnostic Molecular Pathology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan

    Satomi Mukai, Riko Kitazawa, Junko Ishii, Takeshi Kondo, Kiyoshi Mori & Sohei Kitazawa

  2. Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan

    Akihiro Hakozaki & Keisuke Horiuchi

  3. Division of Molecular Pathology, Ehime University Graduate School of Medicine, Ehime, Japan

    Ryuma Haraguch & Sohei Kitazawa

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  1. Satomi Mukai
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  2. Riko Kitazawa
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  3. Junko Ishii
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  4. Takeshi Kondo
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  5. Akihiro Hakozaki
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  9. Sohei Kitazawa
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Correspondence to Sohei Kitazawa.

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Mukai, S., Kitazawa, R., Ishii, J. et al. Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB. Mol Cell Biochem 350, 29–38 (2011). https://doi.org/10.1007/s11010-010-0679-z

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  • DOI: https://doi.org/10.1007/s11010-010-0679-z

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