CDK8 fine-tunes IL-6 transcriptional activities by limiting STAT3 resident time at the gene loci

Abstract

Cytokines are highly pleiotropic ligands that critically contribute to a balanced immune response. We have an incomplete understanding of how cytokines elicit their functional pleiotropy, which has limited their therapeutic use. Here, using Interleukin-6 (IL-6) as a model system, we have performed detailed phosphoproteomic and transcriptomic studies in human Th-1 cells to address the molecular bases defining cytokine functional pleiotropy. We have identified CDK8 as a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses. We found that CDK8 is a major regulator of the IL-6 phosphoproteome and interacts with STAT3 in the nucleus upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecules inhibitors, reduced the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 Ser727 phosphorylation. This, in turn, resulted in retention of tyrosine phosphorylated STAT3 in the nucleus, which increased the binding of STAT3 to target DNA sites in the genome with a concomitant increase in STAT3 mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity via modulation of its gene loci resident time, critically contributing to tuning STAT3 mediated responses.

The CDK8 inhibitor DCA promotes a tolerogenic chemical immunophenotype in CD4+ T cells via a novel CDK8-GATA3-FOXP3 pathway

“…Consistent with this, we demonstrate a previously unappreciated role of STAT1 Ser727 and STAT3 Ser727 phosphorylation in positively regulating Th1 and Th17 differentiation respectively. These findings differ from recent studies suggesting that CDK8 inhibition promotes human Th17 differentiation; possible explanations include differences in species, CDK8 inhibitor and experimental approach (knockin versus transduced allele) ( 26 ). This emphasizes the value of Ser- Ala STAT mutant mice in dissecting (CDK8-related) mechanistic hypotheses, including developing Stat5b Ser730Ala mice to definitively define the role of CDK8-regulated STAT5b Ser730 phosphorylation in T reg differentiation ( 18 ).…”

“…Although the role of Ser727 phosphorylation in Th1/Th17/T reg differentiation is unclear, potential contribution is suggested by the central role of STAT1 Tyr701 and STAT3 Tyr705 tyrosine phosphorylation in Th1 and Th17 differentiation respectively ( 43 - 45 ). Recent studies argue that inhibition of CDK8 promotes Th17 differentiation by attenuating STAT3 Ser727 phosphorylation, emphasizing the importance of investigating this pathway ( 26 ). DCA reduced IL-6-induced STAT3 Ser727 phosphorylation in murine CD4 + T cells, but did not reduce either STAT3 Tyr705 phosphorylation or expression of the hallmark Th17 transcription factor RORγt in cells cultured in Th17 hi conditions; total STAT3 was slightly decreased (Fig.…”

“…Additionally, CDK8 deletion in innate immune NK cells improves tumor surveillance while in adaptive immune cells, CDK8/19 inhibitors promote T reg differentiation ( 18, 22 - 25 ). Recent findings that CDK8 inhibition promotes Th17 differentiation suggest the first pro-inflammatory sequelae ( 26 ). How CDK8 regulates differentiation to other T cell lineages (Th1 and Th2) remains less clear.…”

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex

“…Additionally, the CKM is involved in regulating the pausing of transcription, through phosphorylation and interaction with elongation factors-AFF4, NELF and p-TEFB [5]. Additionally, CDK8 and CDK19 phosphorylate a number of transcription factors at enhancers, such as STAT1/3 [6], SMADs [6], and others, modifying their activity. The fact that functions of the Mediator and its CKM are determined by phosphorylation and mechanical interactions lead to the hypothesis that the Mediator can have kinase-dependent and independent functions, and those inhibitors of CDK8/19 would not always phenocopy the knockouts of Mediator components.…”