Signal Transducer and Activator of Transcription 3, Stat3, has been associated with cytokine-induced proliferation, anti-apoptosis and neoplastic transformation, while constitutively active Stat3 has been found in many human tumors. Stat3 activation by the Src oncoprotein leads to specific gene regulation and the Stat3 mediated signaling pathway is one of the critical signaling pathways involved in Src oncogenesis. Our laboratory demonstrated that engagement of cadherins, which are a class of cell-cell adhesion molecules, can activate Stat3, even in the absence of direct cell to cell contact. Interestingly, a significant increase in total Rac1 and Cdc42 protein levels triggered by cadherin engagement, and an increase in Rac1 and Cdc42 activity, which led to Stat3 activation by a mechanism involving gp130, a common subunit of the IL-6 family of cytokines, was also observed. To investigate the role of gp130 in vSrc transformation, we knocked down gp130 in vSrc transformed cells and found a decrease in the levels of phosphorylated Stat3, the rate of cell migration, rate of cell proliferation and the anchorage-independent growth. It was also previously demonstrated that vSrc had a negative effect on the function of cadherins. Surprisingly, however, despite the fact that vSrc may reduce cadherin adhesion, previous results in our lab showed that cell density still caused a significant increase in Stat3 activity in vSrc transformed cells. Moreover, Stat3 downregulation induced apoptosis in transformed cells which was more pronounced at high cell densities. This may reflect an increased need for Stat3 activity at high densities, possibly to overcome apoptosis, which raised the question of the actual role of cadherins in the density-mediated activation of Stat3 in such cells. The expression of cadherin-11, a type II cell adhesion molecule, is associated with invasive breast cancer and many studies suggest that it may play a significant role in facilitating tumor cell invasion and the formation of metastatic tumors. Since Src and its family members participate in many aspects of tumor progression and metastasis, it was interesting to see if Src needed cadherin-11 for neoplastic transformation. To this effect, when we knocked down cadherin-11 in Balb/c3T3 cells, we observed a significant reduction in levels of phosphorylated Stat3-ptyr705 which was also observed when vSrc was expressed in them. Moreover, expressing vSrc in cells in which cadherin-11 was knocked down also decreased the anchorage –independent growth and increased apoptosis indicating that cadherin-11 is needed for transformation and survival respectively, in vSrc transformed cells. Our results thus demonstrate that cadherin-11 may be a good target for the selective elimination of cells expressing Src and presumably other oncogenes as well. Stat3 activation by cadherins is so potent and important that tumor cell death can be enhanced by cadherin inhibition. In our experiments, the inhibition of cadherin-11 induced apoptosis in Src expressing cells, while the normal cells were not affected. Elucidation of the functions and regulation of cadherin-11 may enhance our understanding of the roles of cadherins in invasive cancer and may provide future targets for therapy. Through our work, the cadherin/IL-6 pathway may emerge as a crucial Stat3 activator in vSrc expressing cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-11-27 18:54:05.777

Background: Proteomic approaches identifying biomarkers have been applied to asthma to only a very limited extent. Methods: With an antibody array (RayBiotech, Norcross, GA, USA), the relative intensity and rank differences of 444 proteins were compared in 24 plasma samples obtained at age 3, 11 from children with and 12 without asthma diagnoses at ages 5 and 9. Protein candidates identified by antibody array were quantitated by ELISA in an enlarged sample. Proteins found to differentiate children with and without asthma were also examined for association with known Year 1 asthma risk factors, eczema, and wheeze. Results: In the antibody array, four proteins had rank differences between asthma and non-asthma groups (FDR < 0.1). By ELISA, mean log (+/- s.e.m.) erythropoietin (EPO) level (IU/l) was lower (0.750 +/- 0.048 vs. 0.898 +/- 0.035; p = 0.006) and mean (+/- s.e.m.) soluble GP130 (sGP130) level (ng/ml) was higher in the asthma vs. the non-asthma group (302 +/- 13 vs. 270 +/- 8; p = 0.041). The other 2 array proteins (galactin-3 and eotaxin-3) did not differ by ELISA by asthma. EPO related to the asthma risk factor, first year eczema, whereas sGP130 related to first year wheeze. Conclusions: Through two independent assessments, age 3 plasma levels of EPO and sGP130 were found related to childhood asthma.

Immune responses during lung infections must be tightly regulated in order to permit pathogen eradication while maintaining organ function. Although mechanisms involve complex networks of cytokines, the interferon (IFN) response has been shown to be an important driver of lung inflammation. Type I IFNs consist of a group of structurally similar cytokines that are produced during virus infection and are an integral part in regulating the immune response. However, in response to certain stimuli, type I IFNs have also been found to be central in the initiation of lung inflammatory responses by inducing the recruitment and activation of immune cells and thus may contribute to disease severity. Another cytokine that has been associated with chronic lung inflammation is the gp130 cytokine, Oncostatin M. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines associated with eosinophilia and alternatively activated (AA/M2) macrophage accumulation. Here we demonstrated that C57Bl/6 mice deficient of the type I interferon receptor (IFNAR1-/-), were less responsive against a suboptimal dose of AdOSM at day 7 post infection compared to AdOSM-treated wild-type. We observed a significant reduction in OSM mRNA and protein levels in AdOSM-treated IFNAR1-/- mice, compared to treated wild-type, which resulted in significant attenuation in OSM-induced inflammatory cell infiltration, epithelial hyperplasia, and alveolar wall thickening. Furthermore, IL-6 overexpression (as a comparator gp130 cytokine), induced lymphocyte accumulation in IFNAR1-/- mice, but at significantly lower levels than AdIL-6-treated wild-type. These results demonstrate that cross talk between IFNAR and gp130 cytokine signaling were required for maximal AdOSM- and AdIL-6-mediated pulmonary inflammation. We also observed that IFNAR1 deficiency directly and negatively regulated OSM-mediated responses in vitro. OSM-induced pSTAT3 levels were consistently lower in murine and human IFNAR1-deficient fibroblasts, compared to OSM-stimulated wild-type cells. This was associated with diminished OSM-induced IL-6 and MCP-1 production from IFNAR1-/- fibroblasts. Furthermore, we found that the combination of OSM and IFN-α led to increased IL-6 production from C57Bl/6 and BALB/c-derived Mouse Lung Fibroblasts (MLFs) then when either cytokine was used alone suggesting that these two cytokines can work in concert. Our findings are the first to suggest that IFNAR signaling participates in OSM-mediated responses in vitro and is required for maximal AdOSM-induced pulmonary inflammation in vivo. / Thesis / Master of Science (MSc)

Stat3 (signal transducer and activator of transcription-3) is activated by a number of receptor and non-receptor tyrosine kinases, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. Results presented in this thesis reveal that Stat3 can also be activated through homophilic interactions by the epithelial (E)-cadherin and cadherin-11, two members of the classical type I and II cadherin family of surface receptors, responsible for the formation of cell to cell junctions. Indeed, by plating cells onto surfaces coated with fragments encompassing the two outermost domains of these cadherins, we definitively demonstrate that cadherin engagement can activate Stat3, even in the absence of direct cell to cell contact. At the same time, levels of the extracellular signal regulated kinase (Erk)1/2, which is often coordinately activated by growth factor receptors and oncogenes, remain unchanged upon cadherin ligation. Most importantly, we report, for the first time, an unexpected surge in total Rac1 and Cdc42 protein levels, triggered by cadherin engagement, and an increase in Rac1 and Cdc42 activity, which is responsible for the Stat3 stimulation observed. Inhibition of cadherin interactions reduced Rac/Cdc42 and Stat3 levels and induced apoptosis, pointing to a significant role of this pathway in cell survival signalling, a finding which could also have important therapeutic implications. To better understand the role of Rac/Cdc42 in the cadherin-mediated Stat3 activation, we compared Stat3 activity in mouse HC11 cells before and after expression of the mutationally activated, RacV12. We demonstrate a dramatic increase in protein levels and activity of both the endogenous Rac and RacV12 with cell density, which was due to inhibition of proteasomal degradation. Moreover, we clearly show that RacV12 expression can activate Stat3 through an increase in expression of members of the IL6 family of cytokines, known potent Stat3 activators. In fact, knockdown experiments indicate that gp130 receptor function, and Stat3 activation, are essential for the migration and proliferation of RacV12-expressing cells, thereby demonstrating that the gp130/Stat3 axis represents an essential target of activated Rac in the regulation of both of these fundamental cellular functions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-02-18 10:38:29.549

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 143-161.

Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.

Vitronectin (VTN) is a glycoprotein in the blood and affects hemostasis. VTN is also present in the extracellular matrix of various organs but little is known about its function in healthy adult tissues. We show, in adult mice, that VTN is uniquely expressed by approximately half of the pericytes of subventricular zone (SVZ) where neurogenesis continues throughout life. Intracerebral VTN antibody injection or VTN knockout reduced neurogenesis as well as expression of pro-neurogenic CNTF, and anti-neurogenic LIF and IL-6. Conversely, injections of VTN, or plasma from VTN+/+, but not VTN−/− mice, increased these cytokines. VTN promoted SVZ neurogenesis when LIF and IL-6 were suppressed by co-administration of a gp130 inhibitor. Unexpectedly, VTN inhibited FAK signaling and VTN−/− mice had increased FAK signaling in the SVZ. Further, an FAK inhibitor or VTN increased CNTF expression, but not in conditional astrocytic FAK knockout mice, suggesting that VTN increases CNTF through FAK inhibition in astrocytes. These results identify a novel role of pericyte-derived VTN in the brain, where it regulates SVZ neurogenesis through co-expression of CNTF, LIF and IL-6. VTN-integrin-FAK and gp130 signaling may provide novel targets to induce neurogenesis for cell replacement therapies.

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