Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--Illinois State University, 1996. / Title from title page screen, viewed May 30, 2006. Dissertation Committee: H. Tak Cheung (chair), Herman E. Brockman, Alan J. Katz, Marjorie A. Jones, Brian J. Wilkinson. Includes bibliographical references (leaves 97-105) and abstract. Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

We have shown herein that β1 integrin stably depleted PC3 sub-clonal cells confer a trend towards increased survival of mice compared to β1 integrin expressing counterparts when tested in an intracardial bone metastasis model. Therefore, we sought to investigate novel factors that mediate β1 integrin-dependent cellular migration and three dimensional growth of prostate cancer PC3 cells in vitro. We show herein that depletion of β1 integrin using siRNA directed techniques results in increased SPARC protein expression. We further show that suppression of SPARC by β1 integrin appears to occur through a JNK dependent mechanism. Moreover, siRNA mediated depletion of β1 integrin results in impaired sphere formation in 3D BME assays. This was mediated in part by the increased production of SPARC. β1 integrin-depleted cells also diminished the enhanced migration of cells on the predominant bone matrix, collagen I. Concomitant SPARC depletion in β1 integrin-depleted cells did not rescue this enhanced migration. These findings suggests that the role of β1 integrin in mediating 3D growth of PC3 cells occurs at least in part through the suppression of SPARC protein expression.

Abstract Type XIII collagen belongs to the group of transmembrane collagens. In this thesis the plasma membrane localisation and function of type XIII collagen have been studied using cell biological methods. Type XIII collagen was found to reside in focal adhesions. It appeared in these structures at a very early stage of their assembly and disappeared from them concurrently with focal adhesion proteins talin and vinculin. Insect cells expressing type XIII collagen showed an enhanced adhesion to certain matrix components. These localisation and adhesion data suggested that the function of type XIII collagen is related to cell adhesion. Supporting this, in tissues type XIII collagen was found to localise to cell-matrix and cell-cell adhesion structures. Type XIII collagen was found to be partly present in cholesterol-enriched membrane microdomains. With other membrane proteins this localisation has been shown to be linked to ectodomain shedding. The connection between the membrane microdomain localisation and the ectodomain shedding of type XIII collagen was also characterised, and it was demonstrated that manipulation of the cellular cholesterol level affected the efficiency of the ectodomain shedding. Additionally, insights into intracellular shedding of type XIII collagen in the Golgi apparatus were obtained. The study of type XIII collagen expression in human cancers revealed that it was enhanced especially in the desmoplastic cancer stroma. Since the increased expression of type XIII collagen was detected during the dysplastic stages, type XIII collagen may be involved in the early pathogenesis of cancer. The result indicated that type XIII collagen is involved in the matrix remodelling. In support of this, the cell culture experiments showed that the soluble type XIII collagen ectodomain altered the vitronectin-rich matrix unfavourable for cell adhesion and spreading. This may enhance cancer metastasis. Type XIII collagen expression was also induced in the remodelled stroma of keratoconus and corneal wounds. Data suggested that myofibroblasts were responsible for the increased expression of type XIII collagen in these situations. Therefore both in cancer and in the corneal pathologies studied, type XIII collagen expression was induced by the activated stromal cells.

Chronic wounds represent a severe socio-economic burden and a key area of unmet clinical need. PKCα is ubiquitous in the skin, particularly the epidermis and functions in numerous pathways that are fundamental to wound repair. By utilising a global PKCα-/- mouse we have identified PKCα-regulated processes both in unwounded skin and during wound healing. PKCα-/- mice display considerably delayed wound healing with a dramatic reduction in re-epithelialisation. By analysing the ultrastructure of the epidermis, I have shown that this delay directly correlates with a failure of wound edge desmosomes to switch to a their adhesive properties. A major risk factor for the development of chronic wounds is age. Crucially, this delay in modulating cell adhesion is conserved in human chronic wounds and aged murine skin. Furthermore, manipulation of PKCα using an inducible bitransgenic mouse containing epidermal specific constitutively active PKCα can accelerate the modulation of desmosome adhesion and subsequently improve re-epithelialisation. Global gene expression analysis of PKCα-/- skin and wounds revealed further defects. Upon wounding, we observed a failure to correctly regulate expression of key collagen and Wnt signalling genes that are essential for correct and timely wound healing. Finally, intrinsic gene expression changes were identified in the skin of PKCα-/- mice, specifically a downregulation of multiple extracellular matrix genes. Of note was the downregulation of small leucine-rich proteoglycans which led to alterations to dermal collagen structure and skin tensile strength. These changes render the PKCα-/- skin susceptible to breaking and wound development. To conclude, we have identified multiple roles for PKCα intrinsically in the skin and also during cutaneous wound healing. Importantly, these intrinsic changes appear to predispose PKCα-/- skin to the development of cutaneous wounds and altered wound-specific processes that manifest in a delayed healing phenotype.

Abstract Type XIII collagen is an integral membrane protein in type II orientation. In cells and tissues type XIII collagen has been located in various adhesive structures, like focal adhesions. Due to this, its biological role has been implicated in cell adhesion. This collagen also exists as a soluble protein due to the release of the ectodomain from the plasma membrane. In this thesis, ectodomain shedding, i.e. enzymatic release of the extracellular domain, was studied in detail, focusing on the phenomenon as it occurs in mammalian cells. It was found that the ectodomain is released by members of the mammalian proprotein convertase family, e.g. furin. Shedding was shown to take place at the cell surface, but based on additional observations, this cleavage may also take place intracellularly in the Golgi apparatus. Various intracellular mechanisms, depending on cell type, were found to be involved in the regulation of ectodomain shedding. Apparently, due to the liberation of the ectodomain, the level of type XIII collagen on the plasma membrane is maintained at a relatively even amount. The released ectodomain was shown to retain biological activity. It showed distinct matrix-specificity so that on vitronectin its influence on cell functions was anti-adhesive, anti-migratory, anti-proliferative and non-supportive of cell spreading. It was also demonstrated to affect the fibronectin matrix assembly in a manner that resulted in reduced amounts of the fibrillar fibronectin matrix. A large collection of human epithelial and mesenchymal cancer samples were screened for type XIII collagen mRNA expression and compared to the expression levels of pre-malignant and normal samples. It was discovered that malignant transformation upregulates the expression of type XIII collagen in mesenchymal cancers and particularly in the stroma of epithelial cancers, more so than in cancer epithelia. TGF-β1 was demonstrated as one factor contributing to the stimulation of expression. Based on cell culture experiments in this study, it was also deduced that the upregulated expression of type XIII collagen and the concomitant shedding of the ectodomain can remodel the tumour stroma, making it inauspicious for adhesion-dependent cell functions, particularly in vitronectin-rich milieu.

Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.

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