Dysregulation of endothelial cell barrier function is associated with a wide variety of human diseases ranging from tumor metastasis to inflammation. The barrier function of endothelial cells is maintained by cell adhesion molecules (CAMs). Immunoglobulin containing and proline-rich receptor-1(IGPR-1) was recently identified as a novel CAM involved in angiogenesis. However, the molecular mechanism of IGPR-1 function in endothelial cells remains largely unknown. The overarching goals of this study were: (A) to determine molecular mechanism by which IGPR-1 stimulates biological responses in cells and (B) to investigate regulation of phosphorylation of IGPR-1 at serine 220 (Ser220), and its role in IGPR-1 function. Our data demonstrate that IGPR-1 undergoes cis-dimerization, which leads to homophilic trans-dimerization of IGPR-1, which is required for its adhesive function. Moreover, we demonstrate that phosphorylation of Ser220 is regulated by trans-dimerization of IGPR-1 and that Glycogen Synthase Kinase 3 (GSK-3) is responsible for its phosphorylation as over-expression of kinase active increased and kinase inactive inhibited phosphorylation of Ser220, respectively. Taken together, the results demonstrate that the coordinated dimerization of IGPR-1 and its homophilic interaction regulates its adhesive function and serine phosphorylation. The adhesive function of IGPR-1 contributes to the barrier function of endothelial cells.

The Signal Transducer and Activator of Transcription-3 (Stat3) is a transcription factor that is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. It was previously demonstrated that cell to cell adhesion causes a dramatic increase in the activity of Stat3 in both normal and tumour cells. This hinted for the first time at the possibility that the role of Stat3 may differ upon cellular confluence. To examine such a mechanism, it is important to evaluate the effect of Stat3 downregulation at different time-points relative to confluence. To examine this, two different approaches for Stat3 downregulation were used: (1) the introduction of high levels of peptidomimetics analogs, which block the Stat3-SH2 domain by using a technique of in situ electroporation. (2) Treatment with two platinum compounds that inhibit Stat3 binding to activated receptors and DNA. The results demonstrated that Stat3 downregulation in vSrc or TAg transformed mouse fibroblast cells or in breast carcinoma lines, induced apoptosis which was more pronounced post-confluence at the time of its peak activity. In contrast, in sparsely growing normal mouse fibroblasts, Stat3 inhibition induced merely a growth retardation. However, in densely growing normal fibroblasts, Stat3 inhibition induced apoptosis. At least in part, apoptosis induced by Stat3 inhibition was mediated by p53, as shown by the resistance to cell death by Stat3 downregulation in colon carcinoma cells, HCT116, where the p53 gene is ablated. Overall, our observations point to the possibility that constitutive activation of Stat3 may lead to tumourigenesis by downregulating wt-53 in cancers that do not have p53 mutations. As a result, targeting Stat3 in cancers with wt-p53 may be a promising therapeutic approach for restoring p53 function, thereby inducing p53-mediated apoptosis. Next, we examined the effect of constitutively activated Stat3 as an oncogene. Stat3C expression in rat F111 fibroblasts induced anchorage independence, but to a lower degree compared to other oncogenes, such as vSrc. Surprisingly Stat3C expression increased gap junction intercellular communication, despite the fact that other oncogenes such as vSrc or vRas effectively block gap junctions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-04-26 01:09:21.654