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Item Genome-wide Analysis Using ChIP-seq Reveals Novel Downstream Targets of Stat3(Office of the Vice Chancellor for Research, 2015-04-17) Corry, Kylie A.; Li, JiliangMany cells are involved in the orchestra that is bone homeostasis--particularly osteoclasts and osteoblasts who 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. Therefore, 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 include employing available ChIP-seq data in order 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 mouse knockout model. A preliminary look at the ChIP-seq data reveals Wnt and Nrf2 signaling to be under the 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.Item Lipopolysaccharides Improve Mesenchymal Stem Cell-Mediated Cardioprotection by MyD88 and stat(Mary Ann Liebert, 2019-04-25) Chu, Xiaona; Xu, Bing; Gao, Hongyu; Li, Bai-Yan; Liu, Yunlong; Reiter, Jill L.; Wang, Yue; Medical and Molecular Genetics, School of MedicineBone marrow-derived mesenchymal stem cells (MSCs) improve cardiac function after ischemia/reperfusion injury, in part, due to the release of cytoprotective paracrine factors. Toll-like receptor 4 (TLR4) is expressed in MSCs and regulates the expression of cytoprotective factors, cytokines, and chemokines. Lipopolysaccharide (LPS) stimulation of TLR4 activates two distinct signaling pathways that are either MyD88 dependent or MyD88 independent/TIR-domain-containing adapter-inducing interferon-β (TRIF) dependent. While it was reported previously that LPS treatment improved MSC-mediated cardioprotection, the mechanism underlying such improved effect remains unknown. To study the role of MyD88 signaling in MSC cardioprotective activity, wild type (WT) and MyD88-/- MSCs were treated with LPS (200 ng/mL) for 24 h. WT and MyD88-/- MSCs with or without LPS pretreatment were infused into the coronary circulation of isolated mouse hearts (Langendorff model) and then subjected to ischemia (25 min) and reperfusion (50 min). Saline served as a negative control. Both untreated and LPS-pretreated WT MSCs significantly improved postischemic recovery of myocardial function of isolated mouse hearts, as evidenced by improved left ventricular developed pressure and ventricular contractility assessment (ie, the rate of left ventricle pressure change over time, ± dp/dt). LPS-pretreated WT MSCs conferred better cardiac function recovery than untreated MSCs; however, such effect of LPS was abolished when using MyD88-/- MSCs. In addition, LPS stimulated stat3 activity in WT MSCs, but not MyD88-/- MSCs. stat3 small interfering RNA abolished the effect of LPS in improving the cardioprotection of WT MSCs. In conclusion, this study demonstrates that LPS improves MSC-mediated cardioprotection by MyD88-dependent activation of stat3.Item Regulation of the germinal center reaction by T helper cells and T regulatory cells(2016-04-11) Wu, Hao; Dent, Alexander L.; Kaplan, Mark H.; Turner, Matthew J.; Zhou, BaohuaGerminal Centers (GCs) are transient lymphoid structures that arise in lymphoid organs in response to T cell-dependent antigen. Within the GC, follicular T helper (TFH) cells promote GC B cell differentiation and in turn the proper antibody production to protect us from invading pathogens. We wished to study the regulation of this process by transcription factors STAT3 and Bcl6. STAT3 is important for both TFH cell differentiation and IL-4 production by Th2 cells. IL-4 is a major functional cytokine produced by TFH cells. To dissect the role of STAT3 in IL-4 production by TFH cells, we generated T cell-specific conditional STAT3 knockout mice (STAT3KO). Compared to WT mice, TFH cell differentiation in STAT3KO mice was partially impaired, both in spleen following sheep red blood cells (SRBC) immunization and in Peyer's patches (PPs). In STAT3KO mice, the numbers of splenic GC B cells were markedly decreased, whereas PP GC B cells developed at normal numbers and IgG1 class switching was greatly increased. Unexpectedly, we found that STAT3 intrinsically suppressed the expression of IL-4 and Bcl6 in TFH cells. Mechanistically, in vitro repression of IL-4 expression in CD4 T cells by Bcl6 required STAT3 function. Apart from TFH cells, the GC reaction is also controlled by regulatory follicular T helper (TFR) cells, a subset of Treg cells. To study the mechanism of how TFR cells regulate the GC reaction, we generated mice specifically lacking TFR cells by specifically deleting Bcl6 in Treg cells. Following immunization, these "Bcl6FC" mice developed normal TFH and GC B cell populations. However, Bcl6FC mice produced altered antigen-specific antibody responses, with reduced titers of IgG and increased IgA. Bcl6FC mice also developed IgG antibodies with significantly decreased avidity to antigen in an HIV-1 gp120 "prime-boost" vaccine model. Additionally, TFH cells from Bcl6FC mice produced higher levels of Interferon-γ, IL-10 and IL-21. Loss of TFR cells therefore leads to highly abnormal TFH and GC B cell responses. Overall, our studies have uncovered unexpected regulatory roles of STAT3 in TFH cell function as well as the novel regulatory roles of TFR cells on cytokine production by TFH cells and on antibody production.Item The role of Stat3 in skeletal development(2017-06-30) Davidson, Rebecca; Li, Jiliang; Yokota, Hiroki; Marrs, KathleenMany factors are present in the development of skeletal tissue. Some factors lead to an increase in bone mass while some lead to a decrease. One factor that is known to have an influence on skeletal development is Signal Transducer and Activator of Transcription 3 (Stat3). This knowledge arose because of a mutation in the Stat3 gene in humans causing a disease called Hyper-IgE Syndrome. This mutation leads to a variety of issues, including decreased bone mass. Because of this, our lab has sought to study Stat3 in its relation to bone. Many studies have already been conducted that discern how Stat3 influences skeletal biology by observing its role in osteoclasts, osteoblasts, and other bone cells. Its role is still unclear, and many studies have provided seemingly contradictory results in how it works on bone tissue. Our lab set up several different studies in order to further elucidate what role Stat3 plays in skeletal development by looking at its effects on osteoblasts and osteoclasts, the bone-forming and bone-destroying cells of the body, respectively. We conditionally knocked out Stat3 in the osteoblasts of mice and compared several different bone parameters to their wild type counterparts at 8 weeks of age. Differences were noted in bone phenotype, including decreased femur length, weight, bone mineral density, and bone mineral content in the cKO compared to their WT counterparts. While no significant difference in trabecular integrity was noted, several differences were observed in cortical bone. These differences indicate that Stat3 has a positive role in osteoblast differentiation, leading to an overall positive effect on bone mass. To observe the role of Stat3 in osteoclasts, in vitro experiments were set up in which pre-osteoclast RAW 264.7 cells were manipulated with Stat3 siRNA or a Stat3 overexpression construct and RANKL to induce differentiation. Using qPCR and western blot assays, it was determined that when Stat3 is knocked down, several important genes in osteoclastogenesis and osteoclast function are more highly expressed than in the control groups. When Stat3 is overexpressed, a similar pattern is observed where these same genes are downregulated in the presence of higher Stat3 levels. These results indicate that Stat3 has an overall inhibitory effect on osteoclastogenesis and osteoclast function, indicating it has a positive effect on bone mass. Future studies could be performed to further elucidate the effects of Stat3 on skeletal development. Isolating the osteoblasts from cKO and WT mice and performing qPCR and western blot assays could be useful in finding out how Stat3 is influencing these cells. Further studies could also be done on the RAW 264.7 cells to find where Stat3 is interacting with the RANKL pathway. A resorption assay could be done with these cells to better understand how function might be influenced by Stat3.