Background: STAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof. Methods: Cultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts. Results: Vitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvβ3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period. Conclusion: These data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

L’aide fournie aux lymphocytes B par les lymphocytes T CD4+ est cruciale pour une réponse humorale de qualité. Les lymphocytes T helper folliculaires (Tfh) jouent un rôle majeur dans l’aide apportée aux lymphocytes B au sein des centres germinatifs et peu de choses sont connues sur la capacité d’aide des autres populations de cellules T CD4+. Le but de notre étude est d’évaluer la capacité d’aide aux lymphocytes B des lymphocytes T helper de type 2 (Th2), une population considérée, à l’origine, comme responsable de l’aide apportée aux lymphocytes B in vivo ainsi que d’analyser le rôle de l’axe IL6 / STAT3 dans la différenciation des lymphocytes Tfh et leur plasticité phénotypique. Nous montrons que les lymphocytes Th2 co-expriment des formes actives des facteurs de transcription STAT6 et STAT3 et que l’expression de STAT3 est requise pour la fonction d’aide aux lymphocytes B des lymphocytes Th2. Nous avons également pu montrer que la voie de signalisation IL-6 / STAT3 durant le développement des lymphocytes Tfh s’oppose à l’expression des gènes associés au programme de différenciation Th2. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Many cells are involved in the orchestra that is bone homeostasis--particularly osteoclasts and osteoblasts, which mediate remodeling of bones. This creates a balance that must be kept in check, otherwise pathologies arise. The JAK-STAT signaling pathway is crucial to maintaining this balance. It has long been known that the transcription factor STAT3 has more profound effects on bone homeostasis than other members of the STAT family of proteins. Recently, a genetic condition called Job’s Syndrome has been specifically linked to point mutations in the Stat3 gene. These patients present with severe bone abnormalities, including prominent foreheads, broad nasal bridges, and abnormal eye spacing. For this reason, our lab has extensively studied conditional knockouts of Stat3 in all three types of bones cells in mice and observed severe deficiencies in numerous parameters of normal bone phenotypes. STAT3 seems to play a principal role in the signaling that takes place upon mechanical loading of bone tissues and calling cells into action where they are needed. Furthermore, STAT3 has been found to be up-regulated in the early-response gene cluster following mechanical loading. Our current approach to studying STAT3’s effects on bone includes both in vivo and in vitro comparisons of WT and KO STAT3 models. The conditional knock-out of STAT3 in 8-week old mice revealed significant phenotypic variations as compared to the WT controls, while no significant differences were observed in cKO newborn pups. We also looked at immortalized WT and STAT3 KO cell lines. The STAT3 KO cells had diminished proliferation rates and decreased differentiation capabilities. Furthermore, STAT3 KO cells showed significantly reduced mRNA levels of both Wnt3a and Wnt5a when exposed to fluid shear stress. By employing available ChIP-seq data, we were able to elucidate the genome-wide binding patterns of STAT3. From the peak distribution, we can begin to uncover novel downstream effectors of STAT3 signaling that are responsible for the observed phenotypes in our conditional knockout mouse model. A preliminary look at the ChIP-seq data reveals Wnt and Nrf2 signaling to be under the putative control of STAT3. In our further research, we endeavor to experimentally confirm the ChIP-seq data for STAT3 with RNA-seq experiments in the hopes of finding potential therapeutic targets for bone pathologies.

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Indiana University-Purdue University Indianapolis (IUPUI) / Inflammatory immune responses are regulated by T cell subsets that secrete specific panels of cytokines. While CD8+ T cells that secrete IFN- and cytotoxic molecules (Tc1 cells) are known to mediate antiviral immunity, IL-17-secreting CD8+ T (Tc17) cells have only recently been described and the development and function of these cells has not been clearly examined. Using in vitro T cell cultures and mice deficient in transcription factors regulating lineage development, we defined Tc17 development and function. Similar to IL-17 secretion from CD4 T cells, IL-17 secretion from Tc17 cells is dependent on the transcription factor Stat3 and inhibited by Stat1. Expression of transcription factors important for Tc1 function, T-bet and Eomesodermin (Eomes), is reduced in Tc17 cells and consistent with this, Tc17 cells are non-cytotoxic in vitro. However, Tc17 cells are unstable and switch to cytotoxic IFN- producing cells when exposed to a Tc1 inducing cytokine, IL-12. Overexpression of the lineage promoting transcription factors T-bet and Eomes is unable to induce a Tc1 phenotype in Tc17 cells and Stat3 is also unable to switch Tc1 cells into Tc17 cells, suggesting additional signals are involved in CD8 T cell lineage commitment. In vivo, Tc17 cells are induced by vaccinia virus, dependant on Stat3, and are capable of mediating antiviral immunity. Tc17 cells acquire an IFN--secreting phenotype after encounter with virus in vivo, however, viral clearance by Tc17 cells is independent of IFN-. Instead, viral clearance is correlated with a gain in T-bet expression and cytotoxic function in Tc17 cells which have encountered virus. The development of anti-viral activity independent of IFN-, suggests that Tc17 cells may mediate anti-viral immunity through novel mechanisms that depend on the ability of Tc17 cells to acquire other phenotypes.

Host innate and adaptive immune responses must be tightly regulated by an intricate balance between positive and negative signals to ensure their appropriate onset and termination while fighting pathogens and avoiding autoimmunity; persistent pathogens may usurp these regulatory machineries to dampen host immune responses for their persistence in vivo. Here, we demonstrate that miR146a is up‐regulated in monocytes from hepatitis C virus (HCV )‐infected individuals compared to control subjects. Interestingly, miR146a expression in monocytes without HCV infection increased, whereas its level in monocytes with HCV infection decreased, following Toll‐like receptor (TLR ) stimulation. This miR146a induction by HCV infection and differential response to TLR stimulation were recapitulated in vitro in monocytes co‐cultured with hepatocytes with or without HCV infection. Importantly, inhibition of miR146a in monocytes from HCV ‐infected patients led to a decrease in IL ‐23, IL ‐10 and TGF ‐β expressions through the induction of suppressor of cytokine signalling 1 (SOCS 1) and the inhibition of signal transducer and activator transcription 3 (STAT 3), and this subsequently resulted in a decrease in regulatory T cells (Tregs) accumulated during HCV infection. These results suggest that miR146a may regulate SOCS 1/STAT 3 and cytokine signalling in monocytes, directing T‐cell differentiation and balancing immune clearance and immune injury during chronic viral infection.

Decades of research have begun to identify oncogenic mut-drivers that are responsible for driving a large percentage of cancers. These high frequency mut-drivers have therapeutics in the clinic for patient treatment. However, there is another group of low frequency mut-drivers that fail to rise above the noise of the high frequently drivers. These low frequency drivers represent a group of genes with untapped potential for new targeted therapies. However, identifying these drivers can be difficult. This study focuses on identifying new functional phosphorylations using the phospho-docking protein 14-3-3. The family of 14-3-3 proteins have been linked to many oncogenic pathways due to the diversity in their client protein interactions. One critical problem in studying 14-3-3 interactors is uncovering the docking site on the phospho-binding partner. Our work indicates that intrinsic disorder and unbiased mass spectrometry identification rate of a given phosphorylation are important for improving the selection of a 14-3-3 docking site. Using a machine learning model, we developed a tool that combines current available 14-3-3 prediction data and our observations about disorder and phosphorylation observation to predict 14-3-3 binding sites. Our publicly available tool "14-3-3-site-finder" produces a rank order list of potential 14-3-3 docking sites that could help overcome the time-consuming process of identifying the correct site. In our efforts of identifying functional phosphorylations with 14-3-3, we have observed that 14-3-3 interacts with a non-receptor tyrosine kinase, TNK1. TNK1 is a poorly characterized kinase that has essentially nothing known about its substrates, function or regulation. TNK1 has been implicated in both tumor suppressor and oncogenic roles. Particularly, a Hodgkin Lymphoma cell line is dependent on a truncated form of TNK1 for growth. Our work uncovers the first mechanism of regulation for this kinase. We found that MARK kinase phosphorylates TNK1 within the proline rich domain allowing 14-3-3 to dock on this phosphorylation. 14-3-3 binding restrains TNK1 in the cytosol and holds TNK1 in an inactive state. Upon the release of 14-3-3, TNK1 moves to a membrane fraction where it is active. One unique feature of TNK1 is that it has a c-terminal ubiquitin association domain (UBA). In vitro ubiquitin pulldowns indicate that the TNK1 UBA has no preference for linkage type or length of ubiquitin. Further, biolayer interferometry indicates that the UBA binds ubiquitin tightly. Mutation of residues within the ubiquitin:TNK1 interface prevent ubiquitin binding and decrease TNK1 activity, preventing downstream oncogenic signaling, suggesting a UBA-centric mechanism of regulation for TNK1. Finally, we developed a small molecule inhibitor, TP-5801, that selectively targets TNK1. TP-5801 prevents downstream TNK1 phosphorylation of STAT3. Further, TP-5801 prolonged the life of mice injected with TNK1 driven Ba/F3 cells. Taken together, our data reveal the first mechanism of kinase regulation for TNK1 involving 14-3-3 binding and ubiquitin association as well as the development of a TNK1 specific therapeutic

Many human tumors harbor persistently-active Signal Transducer and Activator of Transcription (ST AT)3 protein. There is substantial evidence that aberrantly-active STAT3 is a master regulator of events that promote carcinogenesis and human tumor formation. Abnormal STAT3 activity induces uncontrolled growth and survival of cells, thereby contributing to neoplastic transformation and progression. Cisplatin is a major chemotherapeutic modality for ovarian cancer, but is frequently challenged by drug resistance. Given that STAT3 is aberrantly-active in many human tumors, including ovarian cancer, there is the potential that it contributes to the development of Cisplatin resistance, a problem ripe for investigation. This study was conducted to explore the potential that the aberrant STAT3 present in ovarian cancer cells contributes to the decreased sensitivity to Cisplatin observed for ovarian cancer cells. The investigation revealed that STAT3 is aberrantly activated in cancer cell lines resistant to Cisplatin, but not in sensitive cells. Inhibition of aberrant STAT3 activity by the small-molecule STAT3 inhibitor, NSC 74859, increased growth inhibition induced by Cisplatin in resistant ovarian cancer cells. Furthermore, NSC 74859 enhanced apoptosis induced by Cisplatin in resistant cells in vitro by nearly 52%. Collectively, these observations indicate that inhibition of hyperactive STAT3 increases Cisplatin sensitivity, and therefore effectiveness, in resistant cells. Thus, STAT3 represents a viable target for enhancing the sensitivity of ovarian cancer cells to Cisplatin.

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