O015 Unexpected complex STAT3 activation routes and lack of negative feedback inhibition by suppressor of cytokine signaling (SOCS) proteins in Interleukin 23 signal transduction

Signaling of Interleukin 23 (IL-23) via the IL-23 receptor (IL-23R) and the IL-12 receptor beta 1 (IL-12Rβ1) controls innate and adaptive immune responses, and is involved in the proliferation of TH17 cells. Activation of STAT3 appears to be the major signaling pathway of IL-23, and putative STAT binding sites were predicted in the IL-23R but not in the IL-12Rβ1 chain. To elucidate the IL-23 signaling pathway in vitro we generated various expression constructs with amino acid mutations using site-directed mutagensis and deletions within the intracellular domain (ICD) of the murine and human IL-23R. Proliferation of stably transduced Ba/F3-gp130 cells expressing the IL-12Rβ1 and wild-type IL-23R or the mutated/truncated IL-23R variants has been investigated. Phosphorylation and activation of STAT3 was determined by Western blot and FACS analysis. We showed that the predicted STAT binding sites ((p)YXXQ; Y504, Y626 in murine IL 23R and Y484 , Y611 in human IL-23R) mediated STAT3 activation albeit with variable efficiency. Furthermore, we identified two uncommon STAT3 binding/activation sites within the IL-23R. Firstly, the murine IL-23R carried a YPNFQ (Y542) sequence, which acts as an additional activation site of STAT3. Secondly, we identified a phosphotyrosine-independent STAT3 binding motif within the murine and human IL-23R, indicating that pre-association of STAT3 with the IL-23R is required for IL-23 signal transduction. A fourth predicted site, Y416 in murine and Y399 in human IL-23R, was responsible for the activation of the MAPK pathway. In contrast to IL-6-induced short-term STAT3 phosphorylation, cellular activation by IL-23, resulted in long-term STAT3 phosphorylation, indicating that the IL-23R is not a target of negative feedback inhibition by suppressor of cytokine signaling (SOCS) proteins. In summary, the uncommon STAT3 activation routes in combination with the lack of negative feedback inhibition by SOCS proteins of the IL-23R might explain why IL-6 is only a short-term differentiation factor, whereas IL-23 is a long-term proliferation factor of TH17 cells.