Department of Oncology and Diagnostic Sciences, School of Dentistry, University of Maryland, Baltimore, MD 21201, USA.
Cells. 2021 Sep 15;10(9):2432. doi: 10.3390/cells10092432.
Tumor necrosis factor-alpha (TNF-α) signaling regulates phosphorylation of L-plastin, which is involved in forming the nascent sealing zone, a precursor zone for the matured sealing ring. This study aimed to illustrate the molecular mechanisms of L-plastin phosphorylation and the subsequent formation of the nascent sealing zone in osteoclasts treated with TNF-α. Here, we report that anti-TNF-receptor 1, inhibitors of signaling proteins (Src, PI3-K, Rho, and Rho-kinase), and siRNA of TRAF-6 attenuated the phosphorylation of LPL and filamentous actin content significantly in the presence of TNF-α. An inhibitor of integrin αvβ3, PKC, or PKA did not inhibit TNF-α-induced L-plastin phosphorylation. Inhibitors of Src and PI3-K and not Rho or Rho-kinase reduced tyrosine phosphorylation of TRAF-6, suggesting that Src and PI3-K regulate TRAF-6 phosphorylation, and Rho and Rho-kinase are downstream of TRAF-6 regulation. Osteoclasts expressing constitutively active or kinase-defective Src proteins were used to determine the role of Src on L-plastin phosphorylation; similarly, the effect of Rho was confirmed by transducing TAT-fused constitutively active (V14) or dominant-negative (N19) Rho proteins into osteoclasts. Pull-down analysis with glutathione S-transferase-fused SH2 and SH3 domains of Src and PI3-K demonstrated coprecipitation of L-plastin and TRAF-6 with the SH3 and SH2 domains of the PI3-K and Src proteins. However, the actual order of the interaction of proteins requires further elucidation; a comprehensive screening should corroborate the initial findings of protein interactions via the SH2/SH3 domains. Ultimately, inhibition of the interaction of proteins with SH2/SH3 could reduce L-plastin phosphorylation and affect NSZ formation and bone resorption in conditions that display osteoclast activation and bone loss.
肿瘤坏死因子-α（TNF-α）信号转导调节 L-肌动蛋白的磷酸化，该蛋白参与形成新生封闭带，这是成熟封闭环的前体区。本研究旨在阐明 TNF-α处理破骨细胞中 L-肌动蛋白磷酸化和新生封闭带形成的分子机制。在这里，我们报告抗 TNF-α受体 1、信号蛋白（Src、PI3-K、Rho 和 Rho 激酶）抑制剂以及 TRAF-6 的 siRNA 在 TNF-α存在的情况下显著抑制 LPL 的磷酸化和丝状肌动蛋白含量。整合素 αvβ3 的抑制剂、PKC 或 PKA 并不抑制 TNF-α诱导的 L-肌动蛋白磷酸化。Src 和 PI3-K 的抑制剂而非 Rho 或 Rho 激酶减少 TRAF-6 的酪氨酸磷酸化，表明 Src 和 PI3-K 调节 TRAF-6 磷酸化，而 Rho 和 Rho 激酶是 TRAF-6 调节的下游。使用表达组成性激活或激酶缺陷 Src 蛋白的破骨细胞来确定 Src 对 L-肌动蛋白磷酸化的作用；同样，通过将 TAT 融合的组成性激活（V14）或显性负（N19）Rho 蛋白转导到破骨细胞中来证实 Rho 的作用。用 GST 融合的 Src 和 PI3-K 的 SH2 和 SH3 结构域进行下拉分析表明，L-肌动蛋白和 TRAF-6 与 PI3-K 和 Src 蛋白的 SH3 和 SH2 结构域共沉淀。然而，蛋白质相互作用的实际顺序需要进一步阐明；全面筛选应通过 SH2/SH3 结构域证实蛋白质相互作用的初步发现。最终，抑制蛋白质与 SH2/SH3 的相互作用可以减少 L-肌动蛋白磷酸化，并影响在破骨细胞激活和骨质流失的情况下 NSZ 的形成和骨吸收。
