The Bcl6 proto-oncogene, which encodes a transcriptional repressor, is ubiquitously expressed and predominantly in germinal center (GC) B cells. Although the promoter region of the human Bcl6 gene has been reported, enhancer molecules for its high expression in GC B cells were largely unknown. Here we show that transcriptional start sites of the murine Bcl6 gene were different from the reported human one. DNA sequence around the new promoter region is highly conserved between mice and humans and has no canonical TATA or CCAAT box. Two AP-1-binding elements in the promoter region were the major enhancer elements in GC-derived B lymphoma cells, and JunD/AP-1 was detected in GC B cells. In addition, we identified the silencer region with three Bcl6-binding elements around the start site. Bcl6 bound to the silencer elements and its over-expression repressed the promoter activity through the elements. Activated STAT factors (STATs), especially activated STAT3, also bound to the silencer elements in GC B cells and competed with Bcl6 for the binding, suggesting that JunD/AP-1 and activated STATs drive high Bcl6 expression in GC B cells. Since stimulation of splenic B cells with IL-4 or IL-21 induced high Bcl6 expression with induction of junD and activation of STATs, these cytokines may be inducers for its high expression in GC B cells. However, IL-21 but not IL-4 stimulation activated STAT3 in splenic B cells. Thus, IL-21 may be a major inducer for high Bcl6 expression in GC B cells.

Bcl6 functions as a sequence-specific transcriptional repressor, and Bcl6-deficient (Bcl6−/−) mice have been reported to display Th2-type inflammatory diseases in multiple organs. Since IL-18 is a potent stimulator of Th2 cells, we examined the expression of IL-18 mRNA in bone marrow-derived macrophages from Bcl6−/− mice after LPS stimulation. Here we show that the expression was strikingly up-regulated after stimulation. The expression was also up-regulated in RAW264 cells, a murine macrophage cell line, by transfection with the dominant negative type of Bcl6 gene. We identified a putative Bcl6-binding DNA sequence (IL-18BS) upstream of exon 1 of the murine IL-18 gene and three IL-18BSs in the promoter region of human IL-18 gene. Binding of Bcl6 in nuclear protein from resting RAW264 cells to murine IL-18BS was detected by gel retardation assay and chromatin immunoprecipitation assay. The binding activity was diminished gradually in RAW264 cells after LPS stimulation. However, the amount of Bcl6 protein in these cells was constant over the period examined, suggesting the functional modification of Bcl6 protein after stimulation. Furthermore, murine IL-18BS was required for Bcl6 to repress the expression of the luciferase reporter gene under control of the IL-18 promoter. Taken together, Bcl6 is a key regulator of IL-18 production by macrophages.

Mice deficient in the transcriptional repressor B-cell CLL/lymphoma 6 (Bcl6) exhibit similar T helper 2 (TH2) immune responses as patients with allergic diseases. However, the molecular mechanisms underlying Bcl6-directed regulation of TH2 cytokine genes remain unclear. We identified multiple Bcl6/STAT binding sites (BSs) in TH2 cytokine gene loci. We found that Bcl6 is modestly associated with the BSs, and it had no significant effect on cytokine production in newly differentiated TH2 cells. Contrarily, in memory TH2 (mTH2) cells derived from adaptively transferred TH2 effectors, Bcl6 outcompeted STAT5 for binding to TH2 cytokine gene loci, particularlyInterleukin4(Il4) loci, and attenuated GATA binding protein 3 (GATA3) binding to highly conserved intron enhancer regions in mTH2 cells. Bcl6 suppressed cytokine production epigenetically in mTH2 cells to negatively tune histone acetylation at TH2 cytokine gene loci, includingIl4loci. In addition, IL-33, a pro-TH2 cytokine, diminished Bcl6’s association with loci to which GATA3 recruitment was inversely augmented, resulting in altered IL-4, but not IL-5 and IL-13, production in mTH2 cells but no altered production in newly differentiated TH2 cells. Use of a murine asthma model that generates high levels of pro-TH2 cytokines, such as IL-33, suggested that the suppressive function of Bcl6 in mTH2 cells is abolished in severe asthma. These findings indicate a role of the interaction between TH2-promoting factors and Bcl6 in promoting appropriate IL-4 production in mTH2 cells and suggest that chronic allergic diseases involve the TH2-promoting factor-mediated functional breakdown of Bcl6, resulting in allergy exacerbation.

Metformin is a diabetes drug with anticancer properties. Several studies have investigated the effects of metformin combined with chemotherapeutic agents, with controversial results. This study evaluated the efficacy of combined metformin/cisplatin treatment in an endometrial cancer cell line. Ishikawa cells were treated with metformin, cisplatin or both types of treatment. Cell proliferation was evaluated by quantification and colorimetric and thymidine incorporation assays, cell cycle progression was assessed by flow cytometry, and apoptosis by the caspase-3 activity assay. The effects of metformin and cisplatin used in combination were assessed under normoxic (21% O2) and hypoxic (1% O2) conditions. Mitochondrial morphology was examined using the MitoTracker dye, while function was assayed by lactate production. Discrepant results were obtained from the different assays of cell proliferation, with the value obtained from the colorimetric assay being higher than that from cell counts after drug treatment. Combined treatment with metformin (≥2 mM) and cisplatin (1 µM) had additive anti-proliferative effects on cells under normoxic conditions. However, the additive effect of metformin was attenuated under hypoxia. Metformin caused morphological and functional changes in mitochondria, which appeared shortened after exposure to metformin, while the connections between individual mitochondria appeared weaker. Additionally, decreased MitoTracker staining was observed after an 8-h exposure to metformin. The colorimetric assay did not accurately determine the effects of metformin and cisplatin on cell proliferation. The additive effects of metformin on cisplatin-induced inhibition of cell proliferation were attenuated under hypoxic conditions, while metformin compromised mitochondrial structure and function. Additional studies are needed to determine the efficacy of this drug combination in vivo.

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