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Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.

Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein-protein interaction effectively and comprehensively. Biological significance The location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3. With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications.

SRC (steroid receptor co-activator)-1 has been reported to interact with and to be an essential co-activator for several members of the STAT (signal transducer and activator of transcription) family, including STAT3, the major signal transducer of IL (interleukin)-6. We addressed the question of whether SRC-1 is crucial for IL-6- and STAT3-mediated physiological responses such as myeloma cell survival and acute-phase protein induction. In fact, silencing of SRC-1 by RNA interference rapidly induced apoptosis in IL-6-dependent INA-6 human myeloma cells, comparable with what was observed upon silencing of STAT3. Using chromatin immunoprecipitation at STAT3 target regions of various genes, however, we observed constitutive binding of SRC-1 that decreased when INA-6 cells were treated with IL-6. The same held true for STAT3 target genes analysed in HepG2 human hepatocellular carcinoma cells. SRC-1-knockdown studies demonstrated that STAT3-controlled promoters require neither SRC-1 nor the other p160 family members SRC-2 or SRC-3 in HepG2 cells. Furthermore, microarray expression profiling demonstrated that the responsiveness of IL-6 target genes is not affected by SRC-1 silencing. In contrast, co-activators of the CBP [CREB (cAMP-response element-binding protein)-binding protein]/p300 family proved functionally important for the transactivation potential of STAT3 and bound inducibly to STAT3 target regions. This recruitment did not depend on the presence of SRC-1. Altogether, this suggests that functional impairment of STAT3 is not involved in the induction of myeloma cell apoptosis by SRC-1 silencing. We therefore conclude that STAT3 transactivates its target genes by the recruitment of CBP/p300 co-activators and that this process generally does not require the contribution of SRC-1.

Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a major technique for quantitative proteomics using cell culture. It has been applied to the investigation of many different biological processes such diverse as the characterization of signaling pathways and the determination of protein interactions. For investigation of proteins, interacting with the Signal Transducer and activator of transcription 3 (STAT3), we combined SILAC with stringent precipitation of the biotinylated proteins and quantitative mass spectrometry analyses, to detect cellular interaction partners of STAT3 in mammalian cells. STAT3 is a well-known protein, which plays crucial roles in different biological responses including early embryonic development as well as cell growth and apoptosis. Moreover, STAT3 is constitutively activated in oncogene-transformed cells and various primary tumors. Although the JAK-STAT signaling pathway is one of the best known, there appears to be STAT3-specific regulatory proteins, which are not established until now. Hence, finding of novel STAT3-interacting proteins can encourage the knowledge of STAT3-protein network. For metabolic labeling using SILAC, cells were grown in medium containing either only 'light' arginine or only 'heavy' 13C6-arginine, resulting in incorporation of labeled arginine in all sites. In mass spectrometry analyses the peptides containing the 13C6-arginine, has produced a 6 Da difference in mass, relative to the light form. STAT3 fused with a tag for its biotinylation was expressed and biotinylated in HEK-293 cells, which were fully substituted with 13C-labeled arginine in place of the light arginine. In the parallel approach HEK cells, grown in normal medium, were transfected with STAT3 without the tag for biotinylation. After combining the cultured cells, STAT3 and its interacting proteins were specific precipitated, separated by SDS/PAGE, and analyzed by mass spectrometry. This method bears a strong potential to recover now protein interactions.