Macrophages are essential regulators of inflammation and bone loss. RANKL, a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout () rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages (BMDMs) obtained from wildtype (Wt) and animals, and RAW cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger TRAP-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (, , and ). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

中文翻译：

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14-3-3ζ 通过破坏 TRAF6 的稳定性来抑制 RANKL 信号传导


巨噬细胞是炎症和骨质流失的重要调节者。 RANKL 是一种促炎细胞因子，负责巨噬细胞分化为破骨细胞和骨质流失。我们最近发现，14-3-3ze-knockout () 大鼠在炎症性关节炎模型中表现出骨质流失增加。 14-3-3ζ 是一种胞质接头蛋白，积极参与许多信号转导。然而，14-3-3ζ 在 RANKL 信号传导或骨重塑中的作用尚不清楚。我们通过评估 14-3-3ze 在 RANKL 信号传导中的作用，研究了 14-3-3ze 如何影响破骨细胞活性。我们利用从野生型 (Wt) 和动物获得的 14-3-3ζ 缺陷型原代骨髓源性巨噬细胞 (BMDM)，以及使用 CRISPR-Cas9 生成的 RAW 细胞。我们的结果表明，14-3-3ze 缺陷型巨噬细胞在 RANKL 刺激后，具有更大更强的 TRAP 阳性多核细胞，并且骨吸收活性增加。 14-3-3ze 的存在抑制 RANKL 诱导的 MAPK 和 AKT 磷酸化、转录因子（NFATC1 和 p65）核转位以及随后的基因诱导（、、和）。从机制上讲，14-3-3ze 与 TRAF6 相互作用，TRAF6 是 RANKL 受体复合物的重要组成部分。 RANKL 刺激后，14-3-3ze-TRAF6 相互作用增加，而 RANK-TRAF6 相互作用减少。重要的是，14-3-3ze 支持 TRAF6 泛素化和蛋白酶体途径降解，从而抑制下游 RANKL 信号传导。我们共同证明 14-3-3 z 调节 TRAF6 水平以抑制炎症 RANKL 信号传导和破骨细胞活性。据我们所知，这是第一份关于 RANKL 信号传导和破骨细胞激活的 14-3-3ze 调节的报告。