We previously demonstrated that though the human SAA1 gene shows no typical STAT3 response element (STAT3-RE) in its promoter region, STAT3 and the nuclear factor (NF-κB) p65 first form a complex following interleukin IL-1 and IL-6 (IL-1+6) stimulation, after which STAT3 interacts with a region downstream of the NF-κB RE in the SAA1 promoter. In this study, we employed a computational approach based on indirect read outs of protein-DNA contacts to identify a set of candidates for non-consensus STAT3 transcription factor binding sites (TFBSs). The binding of STAT3 to one of the predicted non-consensus TFBSs was experimentally confirmed through a dual luciferase assay and DNA affinity chromatography. The present study defines a novel STAT3 non-consensus TFBS at nt -75/-66 downstream of the NF-κB RE in the SAA1 promoter region that is required for NF-κB p65 and STAT3 to activate SAA1 transcription in human HepG2 liver cells. Our analysis builds upon the current understanding of STAT3 function, suggesting a wider array of mechanisms of STAT3 function in inflammatory response, and provides a useful framework for investigating novel TF-target associations with potential therapeutic implications.

Although radiotherapy improves survival in patients, glioblastoma multiformes (GBMs) tend to relapse with augmented tumor migration and invasion even after ionizing radiation (IR). Aberrant nuclear factor-κB (NF-κB) and signal transducer and activator of transcription factor 3 (Stat3) activation and interaction have been suggested in several human tumors. However, possible NF-κB/Stat3 interaction and the role of Stat3 in maintenance of NF-κB nuclear retention in GBM still remain unknown. Stat3 and NF-κB (p65) physically interact with one another in the nucleus in glioma tumors. Most importantly, glutathione S-transferase pull-down assays identified that Stat3 binds to the p65 transactivation domain and is present in the NF-κB DNA-binding complex. Irradiation significantly elevated nuclear phospho-p65/phospho-Stat3 interaction in correlation with increased intercellular adhesion molecule-1 (ICAM-1) and soluble-ICAM-1 levels, migration and invasion in human glioma xenograft cell lines 4910 and 5310. Chromatin immunopreicipitation and promoter luciferase activity assays confirmed the critical role of adjacent NF-κB (+399) and Stat3 (+479) binding motifs in the proximal intron-1 in elevating IR-induced ICAM-1 expression. Specific inhibition of Stat3 or NF-κB with Stat3.siRNA or JSH-23 severely inhibited IR-induced p65 recruitment onto ICAM-1 intron-1 and suppressed migratory properties in both the cell lines. On the other hand, Stat3C- or IR-induced Stat3 promoter recruitment was significantly decreased in p65-knockdown cells, thereby suggesting the reciprocal regulation between p65 and Stat3. We also observed a significant increase in NF-κB enrichment on ICAM-1 intron-1 and ICAM-1 transactivation in Stat3C overexpressing cells. In in vivo orthotopic experiments, suppression of tumor growth in Stat3.si+IR-treated mice was associated with the inhibition of IR-induced p-p65/p-Stat3 nuclear colocalization and ICAM-1 levels. To our knowledge, this is the first study showing the crucial role of NF-κB/Stat3 nuclear association in IR-induced ICAM-1 regulation and implies that targeting NF-κB/Stat3 interaction may have future therapeutic significance in glioma treatment.

BACKGROUND: The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells.
OBJECTIVE: To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients.
METHODS: We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation.
RESULTS: In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells.
CONCLUSIONS: We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.