Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
Cell Signal. 2024 Dec;124:111424. doi: 10.1016/j.cellsig.2024.111424. Epub 2024 Sep 19.
Inducible nitric oxidase (iNOS) encoded by Nos2 is a representative IFNγ-inducible effector molecule that plays an important role in both innate and adaptive immunity. In the present study, we demonstrated that full-length NF-κB p105 (p105), which is a precursor of NF-κB p50 (p50), is required for full activation of IFNγ-induced iNOS expression in the RAW264.7 mouse macrophage cell line. In comparison to wild-type (WT) RAW264.7 cells, p105 KO RAW264.7 (p105 KO) cells completely lost IFNγ-induced iNOS expression. Despite the limited effect of exogenous expression of p50 in p105 KO cells on IFNγ-induced Nos2 promoter activity, p105 expression fully restored IFNγ-induced Nos2 promoter activity to a level comparable to that of WT cells, suggesting an important role for full-length p105 in IFNγ-induced iNOS expression. While the expression and phosphorylation of JAK1 and STAT1 were rather enhanced in p105 KO cells, the phosphorylation of c-Jun downstream of MAPK signaling was decreased. IFNγ-induced phosphorylation of ERK, a kinase for IFNγ-induced c-Jun phosphorylation, was not significantly reduced in p105 KO cells, although the nuclear activity of ERK was significantly decreased due to its reduced translocation to the nucleus. Expression of iNOS, nuclear translocation of ERK, and phosphorylation of c-Jun were restored by stable supplementation of p105 in p105 KO cells. These results suggest that p105 is required for the nuclear translocation of ERK and the subsequent phosphorylation of c-Jun, which are necessary for full activation of IFNγ-induced iNOS expression. Reduced nuclear translocation of ERK in p105 KO cells was also observed in the activation of ERK following serum starvation, further suggesting that the involvement of p105 in ERK nuclear translocation is not limited to IFNγ-stimulated cells but is a more general function of p105.
诱导型一氧化氮合酶（iNOS）由 Nos2 编码，是 IFNγ 诱导的效应分子的代表，在固有免疫和适应性免疫中都发挥着重要作用。在本研究中，我们证明了 NF-κB p50（p50）的前体全长 NF-κB p105（p105）对于 RAW264.7 小鼠巨噬细胞系中 IFNγ 诱导的 iNOS 表达的完全激活是必需的。与野生型（WT）RAW264.7 细胞相比，p105 KO RAW264.7（p105 KO）细胞完全丧失了 IFNγ 诱导的 iNOS 表达。尽管在 p105 KO 细胞中外源表达 p50 对 IFNγ 诱导的 Nos2 启动子活性的影响有限，但 p105 的表达完全将 IFNγ 诱导的 Nos2 启动子活性恢复到与 WT 细胞相当的水平，表明全长 p105 在 IFNγ 诱导的 iNOS 表达中起重要作用。虽然 p105 KO 细胞中 JAK1 和 STAT1 的表达和磷酸化增强，但 MAPK 信号下游的 c-Jun 磷酸化减少。尽管 IFNγ 诱导的 c-Jun 磷酸化的激酶 ERK 的磷酸化在 p105 KO 细胞中没有明显减少，但由于其向核内的转移减少，ERK 的核内活性显著降低。在 p105 KO 细胞中稳定补充 p105 可恢复 iNOS 的表达、ERK 的核内易位以及 c-Jun 的磷酸化。这些结果表明，p105 对于 ERK 的核内易位和随后的 c-Jun 磷酸化是必需的，这对于 IFNγ 诱导的 iNOS 表达的完全激活是必需的。在血清饥饿后 ERK 的激活中也观察到 p105 KO 细胞中 ERK 的核内易位减少，这进一步表明 p105 参与 ERK 核内易位不仅限于 IFNγ 刺激的细胞，而是 p105 的更普遍功能。
