IL-6 signaling via the STAT3/SOCS3 pathway: Functional analysis of the conserved STAT3 N-domain

The conserved N-domain of the STAT proteins has been implicated in several activities crucial to cytokine signaling including receptor recruitment and STAT activation, cooperative DNA binding and STAT-dependent gene expression. We evaluated the role of the STAT3 N-domain in the IL-6 signal transduction pathway leading to Socs3 gene expression, an essential mechanism that controls the quality and magnitude of IL-6-dependent transcriptional responses. Based on the model for STAT N-domain function in cooperative gene expression and the presence of tandem STAT binding motifs in the murine Socs3 promoter, we anticipated that stabilizing interactions between adjacent STAT3 dimers via N-domain sequences might be essential for Socs3 gene expression. This was underscored by the tight conservation in the location and sequence of the tandem STAT binding sites between the murine and human Socs3 promoters. Using reconstitution into Stat3-/- mouse embryonic fibroblasts (Stat3-/- MEFs), we find that a STAT3 N-domain deletion mutant (Delta 133STAT3) is activated by tyrosine phosphorylation in response to IL-6 and then undergoes dephosphorylation with kinetics similar to full-length STAT3. These results highlight important differences compared to other STATs where the N-domain has been shown to mediate activation (STAT4) or dephosphorylation (STAT1). STAT3 binds predominantly to a single STAT consensus site in the Socs3 promoter, despite the presence of an adjacent STAT motif. Significantly, Delta 133STAT3 stimulates expression of the endogenous Socs3 gene in Stat3-/- MEFs upon IL-6 treatment with an activity similar to reconstituted STAT3, demonstrating that the N-domain is dispensable for Socs3 gene expression. We propose that the Socs3 gene in its chromosomal context is activated by the IL-6/STAT3 pathway independent of STAT3 N-domain sequences.