Department of Hematology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Mol Med Rep. 2017 Dec;16(6):8406-8412. doi: 10.3892/mmr.2017.7631. Epub 2017 Sep 26.
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.
破骨细胞介导的过度骨吸收可能会导致各种溶骨性骨疾病的风险。在本研究中，研究了溴结构域和末端外蛋白抑制剂 I-BET151 对 RAW264.7 细胞破骨细胞生成的影响及其作用机制。将细胞分为 6 组，包括对照组、核因子κB 受体激活剂配体（RANKL）组和另外 4 组，每组均含有不同浓度的 RANKL 和 I-BET151。使用抗酒石酸酸性磷酸酶（TRACP）染色观察 I-BET151 对破骨细胞生成的影响，并计算 TRACP 阳性多核细胞的数量。使用 Western blot 评估肿瘤坏死因子受体相关因子（TRAF6）、激活 T 细胞核因子细胞质 1（NFATcl）、转录因子 p65（p65）、κB 轻多肽基因增强子核因子抑制物-α（IκB-α）、细胞外信号调节激酶、Jun N-末端激酶（JNK）和 p38 的表达。使用逆转录-定量聚合酶链反应（RT-qPCR）测量破骨细胞特异性基因 TRACP、基质金属蛋白酶-9（MMP9）、组织蛋白酶 K（CtsK）和原癌基因酪氨酸蛋白激酶Src（c-Src）的 mRNA 表达水平。TRACP 染色结果表明，I-BET151 抑制 RANKL 诱导的破骨细胞生成，且抑制作用呈剂量依赖性。与 RANKL 组相比，用 I-BET151 处理后，TRACP 多核阳性细胞明显减少。当使用 100 和 200 nM I-BET151 时，对 TRAF6 的抑制作用明显，与 RANKL 组相比，当使用 200 nM 时，NFATcl 明显受到抑制，呈剂量依赖性。I-BET151 可显著抑制所有浓度下 p65 的核转位。I-BET151 还显著抑制 IκB-α 的降解以及 JNK 和 p38 的磷酸化，除了用 50 nM I-BET151 处理后 IκB-α 的表达。RT-qPCR 结果显示，破骨细胞特异性基因 TRACP、MMP9、CtsK 和 c-Src 均被 I-BET151 呈剂量依赖性抑制，除 CtsK 外。综上所述，I-BET151 可能通过 RANKL 信号通路显著抑制 RAW264.7 细胞的破骨细胞生成。
