I‑BET151 inhibits osteoclastogenesis via the RANKL signaling pathway in RAW264.7 macrophages

Cheng,Jing; Zheng,Jifu; Guo,Ninghong; Zi,Fuming

doi:10.3892/mmr.2017.7631

I‑BET151 inhibits osteoclastogenesis via the RANKL signaling pathway in RAW264.7 macrophages

Authors:
- Jing Cheng
- Jifu Zheng
- Ninghong Guo
- Fuming Zi
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Affiliations: Department of Hematology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
Published online on: September 26, 2017 https://doi.org/10.3892/mmr.2017.7631
Pages: 8406-8412

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Abstract

Excessive bone resorption mediated by osteoclasts may lead to the risk of various lytic bone diseases. In the present study, the effects of I‑BET151, a bromodomain and extra terminal domain protein inhibitor, on osteoclastogenesis in RAW264.7 cells and the underlying mechanism of this process was investigated. Cells were divided into 6 groups, including the control group, receptor activator of nuclear factor‑κB ligand (RANKL) group and 4 other groups containing RANKL and I‑BET151 at different concentrations. Tartrate‑resistant acid phosphatase (TRACP) staining was used to observe the effect of I‑BET151 on osteoclastogenesis and the number of TRACP positive multinucleated cells was calculated. Western blotting was used to evaluate the expression of tumor necrosis factor receptor associated factor (TRAF6), nuclear factor of activated T‑cells cytoplasmic 1 (NFATcl), transcription factor p65 (p65), nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitor‑α (IκB‑α), extracellular signal‑regulated kinase, Jun N‑terminal kinase (JNK) and p38. mRNA expression levels of osteoclast specific genes TRACP, matrix metalloproteinase‑9 (MMP9), cathepsin K (CtsK) and proto‑oncogene tyrosine‑protein kinase Src (c‑Src) were measured using the reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). TRACP staining results demonstrated that I‑BET151 inhibited osteoclastogenesis induced by RANKL and the inhibition was dose dependent. TRACP multinucleated positive cells were significantly decreased when treated with I‑BET151 compared with the RANKL group. The inhibitory effect on TRAF6 was significant when concentrations of 100 and 200 nM I‑BET151 were used, and NFATcl was significantly inhibited when a concentration of 200 nM was used compared with the RANKL group, in a dose-dependent manner. Nuclear translocation of p65 was significantly inhibited by I‑BET151 at all concentrations. The degradation of IκB‑α, and phosphorylation of JNK and p38 were also significantly inhibited by I‑BET151, with the exception of the expression of IκB‑α following treatment with 50 nM I‑BET151. The RT‑qPCR results revealed that osteoclast‑specific genes TRACP, MMP9, CtsK and c‑Src were all dose‑dependently inhibited by I‑BET151, except for CtsK. In conclusion, I‑BET151 may significantly suppress the osteoclastogenesis of RAW264.7 cells via the RANKL signaling pathway.

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