FHL2 inhibits the activated osteoclast in a TRAF6-dependent manner
Shuting Bai, … , F. Patrick Ross, Steven L. Teitelbaum
Shuting Bai, … , F. Patrick Ross, Steven L. Teitelbaum
Published October 3, 2005
Citation Information: J Clin Invest. 2005;115(10):2742-2751. https://doi.org/10.1172/JCI24921.
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Research Article Bone biology

FHL2 inhibits the activated osteoclast in a TRAF6-dependent manner

Abstract

TNF receptor–associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-κB (RANK). This event is central to normal osteoclastogenesis. We discovered that TRAF6 also interacts with FHL2 (four and a half LIM domain 2), a LIM domain–only protein that functions as a transcriptional coactivator or corepressor in a cell-type–specific manner. FHL2 mRNA and protein are undetectable in marrow macrophages and increase pari passu with osteoclast differentiation in vitro. FHL2 inhibits TRAF6-induced NF-κB activity in wild-type osteoclast precursors and, in keeping with its role as a suppressor of TRAF6-mediated RANK signaling, TRAF6/RANK association is enhanced in FHL2–/– osteoclasts. FHL2 overexpression delays RANK ligand–induced (RANKL-induced) osteoclast formation and cytoskeletal organization. Interestingly, osteoclast-residing FHL2 is not detectable in naive wild-type mice, in vivo, but is abundant in those treated with RANKL and following induction of inflammatory arthritis. Reflecting increased RANKL sensitivity, osteoclasts generated from FHL2–/– mice reach maturation and optimally organize their cytoskeleton earlier than their wild-type counterparts. As a consequence, FHL2–/– osteoclasts are hyperresorptive, and mice lacking the protein undergo enhanced RANKL and inflammatory arthritis–stimulated bone loss. FHL2 is, therefore, an antiosteoclastogenic molecule exerting its effect by attenuating TRAF6-mediated RANK signaling.

Authors

Shuting Bai, Hideki Kitaura, Haibo Zhao, Ju Chen, Judith M. Müller, Roland Schüle, Bryant Darnay, Deborah V. Novack, F. Patrick Ross, Steven L. Teitelbaum

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FHL2 modulates NF-κB activity. WT and FHL2–/– BMMs were cultured with M-...
FHL2 modulates NF-κB activity. WT and FHL2–/– BMMs were cultured with M-CSF and RANKL. On day 3, the cells were starved for 3 hours and then stimulated with RANKL (100 ng/ml) for up to 2 hours. (A) Nuclear proteins were extracted and NF-κB subunit p65 content was determined by immunoblot. α-Nucleophosmin served as loading control. (B) IκBα and TNF-α mRNA expression was determined by RT-PCR. GAPDH mRNA served as loading control. (C) WT and FHL2–/– BMMs were cultured with M-CSF and RANKL. On day 3, the cells were starved for 3 hours and then stimulated with TNF-α (10 ng/ml) at 0, 5, 15, and 30 minutes. Nuclear proteins were extracted, and NF-κB subunit p65 content was determined by immunoblot. α-Nucleophosmin served as loading control. (D) WT BMMs were retrovirally transduced with empty vector (PMX) or vector expressing FHL2 and selected with blasticidin S, hydrochloride for 3 days. Selected cells were starved for 3 hours and stimulated with RANKL (100 ng/ml) for up to 30 minutes. Phosphorylated (p) and total (T) IκBα were determined by immunoblot. β-actin served as loading control.