Exaggerated inflammatory response results in pathogenesis of various inflammatory diseases. Tumor Necrosis Factor-alpha (TNF) is a multi-functional pro-inflammatory cytokine regulating a wide spectrum of physiological, biological, and cellular processes. TNF induces Focal Adhesion Kinase (FAK) for various activities including induction of pro-inflammatory response. The mechanism of FAK activation by TNF is unknown and the involvement of cell surface integrins in modulating TNF response has not been determined. In the current study, we have identified an oxysterol 25-hydroxycholesterol (25HC) as a soluble extracellular lipid amplifying TNF mediated innate immune pro-inflammatory response. Our results demonstrated that 25HC-integrin-FAK pathway amplifies and optimizes TNF-mediated pro-inflammatory response. 25HC generating enzyme cholesterol 25-hydroxylase (C25H) was induced by TNF via NFκB and MAPK pathways. Specifically, chromatin immunoprecipitation assay identified binding of AP-1 (Activator Protein-1) transcription factor ATF2 (Activating Transcription Factor 2) to the C25H promoter following TNF stimulation. Furthermore, loss of C25H, FAK and α5 integrin expression and inhibition of FAK and α5β1 integrin with inhibitor and blocking antibody, respectively, led to diminished TNF-mediated pro-inflammatory response. Thus, our studies show extracellular 25HC linking TNF pathway with integrin-FAK signaling for optimal pro-inflammatory activity and MAPK/NFκB-C25H-25HC-integrin-FAK signaling network playing an essential role to amplify TNF dependent pro-inflammatory response. Thus, we have identified 25HC as the key factor involved in FAK activation during TNF mediated response and further demonstrated a role of cell surface integrins in positively regulating TNF dependent pro-inflammatory response.

中文翻译：

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肿瘤坏死因子-α 利用 MAPK/NFκB 途径诱导胆固醇-25 羟化酶，通过 25-羟基胆固醇-整合素-FAK 途径放大促炎反应。

过度的炎症反应导致各种炎症性疾病的发病机制。肿瘤坏死因子-α (TNF) 是一种多功能促炎细胞因子，可调节广泛的生理、生物和细胞过程。TNF 诱导粘着斑激酶 (FAK) 进行各种活动，包括诱导促炎反应。TNF 激活 FAK 的机制尚不清楚，并且尚未确定细胞表面整联蛋白参与调节 TNF 反应。在目前的研究中，我们已将氧甾醇 25-羟基胆固醇 (25HC) 鉴定为可溶性细胞外脂质，可放大 TNF 介导的先天免疫促炎反应。我们的结果表明，25HC-integrin-FAK 通路放大和优化了 TNF 介导的促炎反应。TNF 通过 NFκB 和 MAPK 途径诱导产生 25HC 的酶胆固醇 25-羟化酶 (C25H)。具体而言，染色质免疫沉淀分析确定了 AP-1（激活蛋白-1）转录因子 ATF2（激活转录因子 2）在 TNF 刺激后与 C25H 启动子的结合。此外，C25H、FAK 和 α5 整联蛋白表达的丧失以及 FAK 和 α5β1 整联蛋白与抑制剂和阻断抗体的抑制，分别导致 TNF 介导的促炎反应减弱。因此，我们的研究表明，细胞外 25HC 将 TNF 通路与整合素-FAK 信号通路连接起来以获得最佳促炎活性，而 MAPK/NFκB-C25H-25HC-整合素-FAK 信号传导网络在放大 TNF 依赖性促炎反应方面发挥重要作用。因此，