Signal transducer and activator of transcription 1-alpha/beta
nucleus
Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production.
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 Tyr<sup>701</sup> 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 Thr<sup>749</sup>, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr<sup>749</sup> activated the expression of the gene encoding ARID5A, which stabilizes <i>IL6</i> mRNA. Moreover, STAT1 phosphorylated at Thr<sup>749</sup> directly enhanced transcription of the gene encoding IL-12p40 (<i>IL12B</i>). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr<sup>749</sup> facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of <i>ARID5A</i> and <i>IL12B</i> The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr<sup>749</sup> Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both <i>IL12B</i> expression and <i>IL6</i> mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.