E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.
E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression.
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DOI：
10.1016/j.imbio.2014.08.016
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发表时间：
2015-01
期刊：
影响因子：
2.8
通讯作者：
Kornbluth, Jacki
中科院分区：
文献类型：
--
作者：
Lawrence, Donald W.;Gullickson, Gail;Kornbluth, Jacki
受刺激的巨噬细胞在其吞噬体内产生有效的抗微生物活性氧和氮物质。先前的研究表明，E3泛素连接酶自然杀伤细胞溶解相关分子（NKLAM）是一种巨噬细胞吞噬体蛋白，在巨噬细胞抗菌活性中发挥作用。在体内，NKLAM敲除（KO）小鼠在暴露于脂多糖（LPS）时比野生型（WT）小鼠产生更少的一氧化氮（NO）。在体外，我们发现，NO的生产和诱导型一氧化氮合酶（iNOS）蛋白在LPS刺激KO骨髓源性和脾巨噬细胞减少。此外，LPS刺激的NKLAM-KO巨噬细胞显示STAT1酪氨酸磷酸化和干扰素β（IFN β）产生缺陷。JAK/STAT通路对于IFN β的产生至关重要，IFN β增加小鼠中iNOS蛋白的表达。iNOS蛋白的表达也受转录因子NF κ B的调节，因此我们研究了NKLAM是否影响NF κ B的功能。LPS刺激的NKLAM-KO巨噬细胞显示NF κ B亚基p65延迟核转位的证据。这与p65/DNA共定位的减少有关。p65易位缺陷与IKB α降解无关。NKLAM-KO巨噬细胞也表达较少的p65，并显示丝氨酸536处有缺陷的p65磷酸化的证据。重要的是，通过转染荧光素酶报告质粒评估，LPS刺激的NKLAM-KO巨噬细胞具有降低的NF κ B转录活性。总的来说，我们的数据暗示NKLAM作为一种新的巨噬细胞iNOS表达的调节剂。
Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNβ). The JAK/STAT pathway is critical for the production of IFNβ, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.
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