Tissue factor (TF) is a cell-associated receptor for coagulation factor VIIa (FVIIa) that initiates the coagulation cascade and transmits intracellular signals through protease activated receptors (PARs). This thesis documents for the first time that in human glioma cells (U373) oncogenic epidermal growth factor receptor (EGFRvIII) simultaneously upregulates the expression of several elements of the TF pathway (TF, FVIIa, PAR-1 and PAR-2). In the absence of EGFRvIII, TF triggers tumor formation, albeit with a long latency, while treatment of glioma cells with FVIIa activates MAPK phosphorylation and stimulates the expression of angiogenic factors (VEGF and IL-8). Moreover, selective targeting of the host (mouse) TF reveals its independent role in glioma tumorigenesis. We propose that TF may represent an attractive potential target to treat human brain tumors.

Oocyte maturation and ovulation are two major events that occur in fish prior to spawning. While earlier studies have shown that 17α, 20β-dihydroxy-4-pregnen-3-one (17,20β-P) and the insulin-like growth factor (IGF) system are regulators of oocyte maturation in the zebrafish (Danio rerio), it is not known whether these hormones play a role in regulating prostaglandin synthesis which is thought to mediate ovulation. I determined if 17,20β-P and human IGF-1 affect the expression of genes involved in prostaglandin biosynthesis including phospholipase A2 (cpla2) and cyclooxygenase-1/2 (ptgs1/ptgs2), or prostaglandin F2α (PGF2α) levels. 17,20β-P and IGF-1 stimulated oocyte maturation in mid-vitellogenic (MV) and full grown (FG) follicles. In FG follicles, 17,20β-P increased cpla2 expression, whereas IGF-1 increased cpla2 and ptgs2 expression. Both 17,20β-P and IGF-1 increased PGF2α production. The phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling pathways were shown to mediate IGF-1- and 17,20β-P-induced oocyte maturation and cpla2 and ptgs2 expression. Collectively, these results demonstrate that 17,20β-P and IGFs are important regulators of oocyte maturation and prostaglandin synthesis in zebrafish.

Angiogenesis is the formation of new blood vessels by sprouting from pre-existing ones. Transforming growth factor-beta (TGFβ) promotes angiogenesis and is a known inducer of endothelial-mesenchymal transition (EndMT), a process whereby endothelial cells become fibroblastic and motile. We hypothesize that TGFβ-induced EndMT enables endothelial cells to detach from the mature vessel and migrate to form the sprout that becomes a new vessel during angiogenesis. This study characterized EndMT in response to TGFβ +/- vascular endothelial growth factor (VEGF). Bovine aortic endothelial cells (BAEC) were stimulated with TGFβ +/- VEGF for prolonged periods. Confocal imaging and immunoblotting analyses revealed the strongest EndMT response at 5 ng/ml of TGFβ after 144 hours of exposure. A three-dimensional collagen model of angiogenesis revealed a potential relationship between EndMT and blood vessel sprouting. These results suggest that EndMT induction in BAECs requires high concentrations and prolonged exposure to TGFβ and is not significantly influenced by VEGF. / NSERC

Transforming growth factor-beta (TGFβ) is an important signaling molecule that regulates several cellular processes including angiogenesis. However, its effects on angiogenesis are complex, with it being pro-angiogenic only at low concentrations (Pepper et al., 1993). Evidence suggests that downstream signaling pathways of TGFβ may be activated in a dose-dependent fashion. In fact, previous work in our laboratory has shown that the non-canonical Par6 polarity pathway gets preferentially activated at low concentrations. Considering the different cellular effects of downstream signaling pathways, we propose that TGFβ may modulate its effects on angiogenesis via differential activation of the canonical Smad and non-canonical Akt, FAK, NFκB and Par6 polarity signaling pathways. Based on this premise, bovine aortic endothelial cells were treated with TGFβ1 and TGFβ2 at concentrations ranging from 0.05 to 5 ng/mL. The activation patterns of canonical and non-canonical signaling pathways were studied via western blotting; with the use of phospho-specific antibodies against Smad2, Akt, FAK and NFκB. Preliminary results reveal that high concentrations of both TGFβ1 and TGFβ2 (5 ng/mL) cause preferential activation of Smad2, while the Akt, FAK and NFκB signaling pathways do not appear to become activated in response to TGFβ1 or TGFβ2 at the concentrations and time points studied. These results suggest the effect of TGFβ on angiogenesis may not involve Akt, FAK or NFκB signaling, but may involve dose-dependent signaling of the Smad signaling pathway. / NSERC

The survival of breast cancer patients declines when tumors are invasive and have an increased possibility of metastasizing to distal sites. Transforming Growth Factor-beta (TGF-beta) suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells. However, at late stage of mammary carcinogenesis, due to genetic and epigenetic alterations, TGF-beta loses its cytostatic actions, and contributes to tumor invasion by promoting cell proliferation, Actin cytoskeletal reorganization, as well as Epithelial to Mesenchymal Transition (EMT). Despite the key role of TGF-beta1 in tumor suppression as well as tumor progression, the molecular mechanisms underlying the conversion of TGF-beta form an inhibitor of proliferation in mammary breast cancer cells to an inducer of their cell growth and EMT have not been fully elucidated. Thus, acquiring a basic knowledge on the mechanism of TGF-beta regulating its target genes and its contribution to cancer progression may highlight new avenues for cancer therapy development. This prompted us to further investigate and identify TGF-beta-inducible genes that may be involved in TGF-beta biological responses during tumorigenesis. / In this thesis, we identified Talin as a novel TGF-beta1 target gene that acts as an antagonist to inhibit TGF-beta-mediated cell growth arrest and transcriptional activity in mammary cancer cell line, MCF-7. Searching for new partners of activated Smads, we found that TGF-beta1 induces Talin translocation from cytosol to the plasma membrane where Talin physically interacts with the TGF-beta1 signaling components, the Smads and the receptors. Furthermore, we observed that TGF-beta1 stimulation leads to the formation of Actin stress fibers where Talin was detected at the end of these stress fibers. Taken all together, the obtained data show that TGF-beta1 positively induced expression of Talin and suggests a role for Talin, which acts as a negative feedback loop to control TGF-beta biological responses.

Grb10 is a member of the Grb7 family of adaptor proteins that also includes Grb7 and Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of Grb10 to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as well as many non-receptor molecules such as MEK1, Raf-1, and Nedd4. Grb10 has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-1 and the mitochondrial membrane. / In this report the pattern of transient Grb10 translocation following IGF-I cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new Grb10 binding partners.

The problems and high medical costs associated with chronic wounds necessitate an economical bioactive wound dressing. A new strategy was investigated to inhibit MMP-9 proteases and to release epidermal growth factor (EGF) to enhance healing. Doxycycline (DOX) and EGF were encapsulated on polyacrylic acid modified polyurethane film (PAA-PU) using Layer-by-Layer (LbL) assembly. The number of bilayers tuned the concentration of DOX and EGF released over time with over 94% bioactivity of EGF retained over 4 days. A simple wound model in which MMP-9 proteases were added to cell culture containing fibroblast cells demonstrated that DOX inhibited the proteases providing a protective environment for the released EGF to stimulate cell migration and proliferation at a faster healing rate. In the presence of DOX, only small amounts of the highly bioactive EGF are sufficient to close the wound. Results show that this is new and promising bioactive dressing for effective wound management.

The aim of this work was to investigate the role of TGFbeta family members in the induction of cell growth arrest and apoptosis in immune cell types. The TGFbeta superfamily is a large group of evolutionary conserved polypeptide growth factors, involved in different physiological processes. Any deregulation of the different components of the TGFbeta signaling pathway, has been largely implicated in multiple human critical disorders including cancer. Activin, originally isolated from gonadal fluid, and more recently described as an antiproliferative and proapoptotic factor in different cell types has been implicated in different immune functions. In particular, activin and TGFbeta play an important role in the haematopoietic tissue. They are critical death inducers in the immune system contributing to the elimination of different activated immune cell types. Control of immune cell proliferation, activation and subsequent elimination of activated cell populations by cell growth arrest and apoptosis are critical events for controlling infections and preventing autoimmune disease. However, very limited information about the downstream target genes and their signaling mechanism that relay on the inhibitory effects on cell growth by activin and TGFbeta ligands. Using a screen for genes that are differentially regulated by activin and TGFbeta in haematopoietic cells, we found that the phospholipids phosphatase SHIP-1 was strongly upregulated by activin and TGFbeta. Thus, we hypothesized that TGFbeta and activin induce cell growth arrest in immune cells through up-regulation of SHIP-1 with a significant and subsequent decrease in PtdIns 3,4,5-P3 levels affecting cell survival. Furthermore, we attempted to characterize the different intracellular signalling pathways downstream of these serine/threonine kinase receptors that lead to SHIP-1 overexpression as well as the transcription factors involved in the mediation of transcriptional regulation of the SHIP-1 gene promoter. / Chapter 1 provides a broad introduction to the field of TGFbeta signaling focusing on TGFbeta-induced apoptosis in immune cell types and the biology of inositol phosphatases involved in phospholipide metabolism, mainly focused on SHIP-1. Chapter 2 contains data demonstrating that the activin/TGFbeta-induced cell growth arrests and apoptosis through expression of SHIP-1. Data in chapter 3 proved evidences about the cross-talk between the Smad and JNK MAP kinase signalling pathways and their role in the transcriptional regulation of the SHIP-1 gene promoter. Finally, chapter 4, is focused on the discussion and the propose model of how activin/TGFbeta-induced SHIP-1 expression blocks induction of cell survival signals. As a dynamic cellular and molecular process the induction of SHIP-1 by TGFbeta ligands might be in co-association with other apoptosis molecules leading to cell growth arrest and apoptosis in different cell populations of the immune system.

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Chondroitin sulphate proteoglycans (CSPGs) from the glial scar inhibit axonal regeneration following spinal cord injury. CSPG expression can be induced by transforming growth factor β (TGFβ), which suggests that inhibition of TGFβ may reduce CSPG levels. Astrocytes were treated with cyclic AMP (cAMP), which reduced TGFβ signaling protein Smad2 in astrocytes. However, cAMP-treated astrocytes showed strong neurocan expression following TGFβ treatment, which suggests that TGFβ may mediate CSPG expression through non-Smad pathways. Smad2 or Smad4 were knocked down in astrocytes using siRNA and TGFβ-induced neurocan, brevican and aggrecan expression were still observed, indicating that Smad signaling is not required for CSPG expression. Administration of a PI3K/Akt inhibitor produced significant reductions in neurocan, brevican and aggrecan expression in astrocytes, which suggests that PI3K/Akt pathway mediates CSPG expression. Erk1/2 inhibitor treatment did not reduce CSPG expression significantly. Targeting non-Smad signaling pathways may therefore be effective strategies to reduce CSPG expression following injury.