Interleukin-6-dependent survival of multiple myeloma cells involves the Stat3-mediated induction of micro-RNA-21 through a highly conserved enhancer

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

Interleukin 6 increases production of cytokines by colonic innate lymphoid cells in mice and patients with chronic intestinal inflammation

Background & Aims: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods: ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. Conclusions: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells

MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4

MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. It is significantly elevated in the majority of human tumors and functionally linked to cellular proliferation, survival and migration. In this study, we used two experimental-based strategies to search for novel miR-21 targets. On the one hand, we performed a proteomic approach using two-dimensional differential gel electrophoresis (2D-DIGE) to identify proteins suppressed upon enhanced miR-21 expression in LNCaP human prostate carcinoma cells. The tumor suppressor acidic nuclear phosphoprotein 32 family, member A (ANP32A) (alias pp32 or LANP) emerged as the most strongly downregulated protein. On the other hand, we applied a mathematical approach to select correlated gene sets that are negatively correlated with primary-miR-21 (pri-miR-21) expression in published transcriptome data from 114 B-cell lymphoma cases. Among these candidates, we found tumor suppressor SMARCA4 (alias BRG1) together with the already validated miR-21 target, PDCD4. ANP32A and SMARCA4, which are both involved in chromatin remodeling processes, were confirmed as direct miR-21 targets by immunoblot analysis and reporter gene assays. Furthermore, knock down of ANP32A mimicked the effect of enforced miR-21 expression by enhancing LNCaP cell viability, whereas overexpression of ANP32A in the presence of high miR-21 levels abrogated the miR-21-mediated effect. In A172 glioblastoma cells, enhanced ANP32A expression compensated for the effects of anti-miR-21 treatment on cell viability and apoptosis. In addition, miR-21 expression clearly increased the invasiveness of LNCaP cells, an effect also seen in part upon downregulation of ANP32A. In conclusion, these results suggest that downregulation of ANP32A contributes to the oncogenic function of miR-21

Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells.

Signal transducer and activator of transcription 3 (Stat3) dimerization is commonly thought to be triggered by its tyrosine phosphorylation in response to interleukin-6 (IL-6) or other cytokines. Accumulating evidence from in vitro studies, however, suggests that cytoplasmic Stat3 may be associated with high-molecular-mass protein complexes and/or dimerize prior to its activation. To directly study Stat3 dimerization and subcellular localization upon cytokine stimulation, we used live-cell fluorescence spectroscopy and imaging microscopy combined with fluorescence resonance energy transfer (FRET). Stat3 fusion proteins with spectral variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were constructed and expressed in human hepatoma cells (HepG2) and human embryonic kidney cells (HEK-293). Like wild-type Stat3, the fusion proteins redistributed from a preferentially cytoplasmic to nuclear localization upon IL-6 stimulation and supported IL-6-dependent target gene expression. FRET studies in cells co-expressing Stat3-CFP and Stat3-YFP demonstrated that Stat3 dimers exist in the absence of tyrosine phosphorylation. IL-6 induced a 2-fold increase of this basal FRET signal, indicating that tyrosine phosphorylation either increases the dimer/monomer ratio of Stat3 or induces a conformational change of the dimer yielding a higher FRET efficiency. Studies using a mutated Stat3 with a non-functional src-homology 2 (SH2) domain showed that the SH2 domain is essential for dimer formation of phosphorylated as well as non-phosphorylated Stat3. Furthermore, our data show that visualization of normalized FRET signals allow insights into the spatiotemporal dynamics of Stat3 signal transduction

Analysis of the STAT3 interactome using in-situ biotinylation and SILAC

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

Interleukin-6-dependent survival of multiple myeloma cells involves the Stat3-mediated induction of micro-RNA-21 through a highly conserved enhancer

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