The lipopolysaccharide (LPS)–induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-β (IFN-β), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr⁷⁰¹. Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-β signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr⁷⁴⁹, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr⁷⁴⁹ activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr⁷⁴⁹ directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr⁷⁴⁹ facilitated the binding of STAT1 to a noncanonical DNA motif (5′-TTTGANNC-3′) in the promoter regions of ARID5A and IL12B. The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr⁷⁴⁹. Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.

脂多糖 (LPS) 诱导的 Toll 样受体 4 (TLR4) 内吞作用是产生干扰素-β (IFN-β) 的重要步骤，IFN-β 通过在 Tyr ⁷⁰¹磷酸化的 STAT1 激活抗病毒反应基因的转录。在这里，我们表明 STAT1 独立于 IFN-β 信号传导和关键的促炎调节因子 NF-κB 调节 TLR4 内吞下游的促炎细胞因子的产生。在人类巨噬细胞中，TLR4 内吞作用在 Thr ⁷⁴⁹处激活了 STAT1 的非经典磷酸化，随后通过不同的机制促进了白细胞介素 6 (IL-6) 和 IL-12p40 的产生。在 Thr ⁷⁴⁹磷酸化的 STAT1激活了编码 ARID5A 的基因的表达，从而稳定了IL6mRNA。此外，在 Thr ⁷⁴⁹磷酸化的 STAT1直接增强了编码 IL-12p40 ( IL12B )的基因的转录。与影响 STAT1 核易位不同，Thr ^{749 的}磷酸化促进了 STAT1 与ARID5A和IL12B启动子区域中非经典 DNA 基序（5'-TTTGANNC-3'）的结合。TLR4 的内吞作用诱导激酶 TBK1 和 IKKβ 之间形成复合物，其介导 STAT1 在 Thr ⁷⁴⁹处的磷酸化。我们的数据表明，响应 LPS 的非经典磷酸化赋予 STAT1 不同的 DNA 结合和基因调节特性，促进IL12B表达和IL6mRNA 稳定。因此，我们的研究为 TLR4 内吞作用如何调节促炎细胞因子的产生提供了一个潜在的机制。