Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFκB

Signal transducer and activator of transcription 3 (STAT3), one of seven STAT family members, is activated in response to interleukin-6 (IL-6) (Akira et al. 1994). Many cytokines use the common gp130 receptor to activate the phosphorylation of STAT3 on tyrosine residue 705, leading to the formation of dimers through reciprocal phosphotyrosine–SH2 domain interactions. Several growth factors also stimulate STAT3 activation. STAT3 dimers bind to specific γ-interferon activation sequence (GAS) elements (TTCNNNGAA) in the promoters of the induced genes (Seidel et al. 1995). Constitutive activation of STAT3 is observed in many types of tumors. Thus, STAT3 is an oncogene, promoting cell proliferation and survival (Haura et al. 2005; Hodge et al. 2005). STAT3 is persistently phosphorylated in many human cancer cell lines and primary tumors, including hepatocellular carcinomas, breast cancers, prostate cancers, and head and neck cancers, and also in several hematological malignancies. Furthermore, STAT3 is necessary for v-src-induced transformation, and a constitutively active mutant of STAT3 can transform fibroblasts in cooperation with other transcription factors (Joo et al. 2004). Genes encoding proteins that regulate cell survival, including Bcl-2, Bcl-xL, mcl-1, and Fas, are direct targets of STAT3, as are genes encoding the cell cycle regulatory proteins cyclin D1, cyclin E1, and p21. In addition, other transcription factors, including c-Myc, c-Jun, and c-Fos, are themselves STAT3 targets (Hirano et al. 2000). STAT3 also functions as a transcriptional repressor of p53 expression: Blocking STAT3 in cancer cells up-regulates the expression of p53, leading to p53-mediated apoptosis (Niu et al. 2005). Major mechanisms of STAT3 activation in tumor cells are autocrine production of IL-6 and paracrine activation by IL-6 from stroma and infiltrating inflammatory cells. Indeed, circulating IL-6 levels are usually high in cancer patients (Giannitrapani et al. 2002). STAT3 activation provides an important link between inflammation and cancer. For example, Tebbutt et al. (2002) generated gp130757F/F mice, which carry a Y757F point mutation that disrupts the binding of the negative regulators SOCS3 and SHP2 to gp130. As a result, these mice show hyperactivation of STAT3, resulting in chronic gastric inflammation and distal stomach tumors. There are several reports that STAT3 and NF-κB interact with each other (Battle and Frank 2002). For example, Hagihara et al. (2005) demonstrated that STAT3 forms a complex with the p65 subunit of NFκB following stimulation of cells with IL-1 plus IL-6, and that the bound STAT3 interacts with nonconsensus sequences near the κB element of the SAA promoter. Moreover, they showed that a complex that includes STAT3, p65, and p300 is essential for the synergistic induction of the SAA gene by IL-1 plus IL-6. Yu et al. (2002) reported a physical and functional interaction between STAT3 and p65 that inhibits transcriptional activation of the iNOS gene. Yoshida et al. (2004) showed that STAT3 and p65 physically interact in vivo and that p65 homodimers can cooperate with unphosphorylated STAT3 (U-STAT3) when bound to a specific type of κB motif. Reciprocally, this interaction appears to inhibit the function of GAS-binding sites for STAT3. In contrast, the p50 subunit of NFκB can cooperate with phosphorylated STAT3 (P-STAT3) bound to GAS sites (Yoshida et al. 2004). Previous work from this laboratory has shown that STAT1 can drive gene expression even in the absence of tyrosine phosphorylation. For example, Chatterjee-Kishore et al. (2000) showed that unphosphorylated STAT1 binds to IRF1, forming a complex that activates a half GAS-half ICS element in the LMP2 promoter. In the case of STAT3, our recent work (Yang et al. 2005) showed that its high-level expression drives the transcription of many genes in the complete absence of tyrosine phosphorylation, a function quite distinct from the role of P-STAT3 in driving inducible gene expression. This activity of STAT3 is likely to have important physiological functions, since the STAT3 gene has a GAS element that drives a major increase in the concentration of STAT3 protein in response to signal-dependent tyrosine phosphorylation of STAT3 (Kojima et al. 1996; Narimatsu et al. 2001). We now address the mechanism through which U-STAT3 regulates gene expression, showing that, for many genes, it does so through its ability to interact with NFκB. To understand how U-STAT3 functions, we expressed the Y705F mutant of STAT3, which cannot be phosphorylated on residue 705, at a high level in untransformed human mammary epithelial hTERT-HME1 cells and used coimmunoprecipitation and chromatin immunoprecipitation (ChIP) assays to identify cofactors and DNA elements to which Y705F-STAT3 binds. Our data reveal that Y705F-STAT3 forms a complex with unphosphorylated NFκB (U-NFκB), binding to the κB elements of promoters, such as that of the RANTES (CCL5) gene, to induce their transcription.