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 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, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr activated the expression of the gene encoding ARID5A, which stabilizes mRNA. Moreover, STAT1 phosphorylated at Thr directly enhanced transcription of the gene encoding IL-12p40 (). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of and The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both expression and mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.