The passage of fluid, ions and macromolecules across the epithelium is controlled primarily by epithelial tight junctions. Altered epithelial permeability is associated with lung disease, and barrier function is impaired by the Th2 cytokine IL-13. This thesis investigates the signalling pathways involved in the modulation of the epithelial barrier by IL-13 stimulation. Initial experiments demonstrated that the human sub-bronchial epithelial cell line Calu-3 could be easily manipulated when grown using an air-liquid culture system. Expression of various key tight junction proteins was demonstrated, as well as a high trans-epithelial resistance (TER) for up to 7 days. Stimulation with IL-13 resulted in a decrease in TER compared with controls and this decrease was shown to be prevented with the PI3K inhibitor ZSTK474. IL-13 did not increase paracellular permeability of the epithelial monolayer to FITC-dextran from the apical to the basolateral chamber and ZSTK474 did not influence FITC-dextran flux. Immunocytochemistry showed that the expression of the tight junction protein claudin 2 was increased by IL-13 stimulation and this change in expression was shown to be PI3K dependent with the PI3K inhibitor ZSKT474 preventing the increase. Further studies were carried out in an attempt to uncover the PI3K isoform responsible for the effects seen on both the TER and the TJ expression. It was shown that inhibition of the p110α isoform with PIK75 mimicked the result observed with the pan-PI3K inhibitor ZSTK474 and prevented the IL-13-induced claudin 2 upregulation. However none of the PI3K isoform inhibitiors showed the prevention of TER, as shown by the pan PI3K inhibitor ZSTK474. The role of STAT6 in TJ modulation was shown to be similar to that of PI3K, in that inhibition of STAT6 had a positive effect on the epithelial barrier by preventing the IL-13-induced TER decrease and the increase in the expression of claudin 2. In addition, both PI3K inhibition and STAT6 inhibition demonstrated effects on basal TER and claudin 2 expression, indicating that both pathways are involved in maintenance of epithelial barrier integrity.

Forming and maintaining an intact epidermal permeability barrier (EPB) is necessary to mammalian health and dysregulation of this process can result in serious complications. Tight junctions (TJs) and their integral proteins the Claudins (Cldns) have both structural and signaling importance to the skin barrier and the latter is most likely mediated via Cldn tail interaction with cytoplasmic proteins. Given that the family member Cldn6 is known to be important to EPB function, we set out to determine the contribution of its cytoplasmic tail domain to TJ-mediated homoeostasis. Using transgenic mouse models, we overexpressed epidermal-targeted tail truncation mutants and assessed EPB formation and maintenance. We then used yeast 2-hybrid and quantitative proteomic approaches to identify proteins that interact with this tail region and to assess the downstream effects of overexpressing these proteins in human keratinocytes in culture. We demonstrate that a 10 amino acid region in the cytoplasmic tail is required for efficient epidermal maturation and injury repair and that our mouse models may be applicable to postnatal epidermal maturation and human skin aging studies. We show that in addition to the known interacting partner ZO1, the C-terminal tail of Cldn6 also binds FIZ1 (Flt3 interacting zinc finger protein-1), which we characterize for the first time as a mitogenic factor for keratinocytes. FIZ1 stimulates autocrine pathways involving secreted heparin-binding factors IGFBP3 and DKK1, sensitization to IGF signaling, MAP/ERK activation and increased G1 progression. Specific transcription factors, protein kinases and signaling scaffolds that we identified as novel FIZ1-binding partners likely mediate this signaling. Our studies on the Cldn6 cytoplasmic tail support the importance of this region for epidermal maturation and for maintenance of skin homeostasis throughout life. They also delineate the potential for tail interactors such as ZO1 and FIZ1 to act in concert with Cldns in TJ-based signaling networks to regulate the balance between proliferation and differentiation in keratinocytes. These findings provide new insight into the role of the Cldn6 cytoplasmic tail and will ultimately aid in the development of new diagnostic tools and therapeutic approaches for the treatment of skin conditions rooted in barrier defects.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease in premature babies. Despite significant morbidity and mortality, the cause of this disease remains unclear and there are no preventative treatments available. Prematurity and enteral feeding of infant formula are considered to be the primary risk factors for development of NEC. Interestingly, the incidence of NEC is six to ten times lower in breast-fed babies compared to those that were formula-fed. The factors responsible for the protective effect of breast milk against NEC have not been identified, but epidermal growth factor (EGF) is one of the most promising candidates. EGF is found at high concentrations in human milk, but is not present in any commercial formula. Mothers with extremely premature babies have 50-80% higher levels of EGF in their breast milk compared to mothers with full term infants. This suggests that EGF plays an important role in the development of premature infants. Our studies have shown that supplementation of EGF into formula significantly reduces the incidence of NEC in a neonatal rat model. However, the mechanisms underlying this EGF-mediated reduction of NEC are not understood. The overall hypothesis of this dissertation is that the protective effect of EGF in NEC pathogenesis is mediated via increased expression of pro-survival genes and strengthening of the mucosal barrier. The results of the studies within this dissertation demonstrate that treatment with EGF significantly decreases intestinal epithelial cell apoptosis at the site of NEC injury by up-regulating anti-apoptotic genes and down-regulating pro-apoptotic genes. Furthermore, supplementation of formula with EGF strengthens the mucosal barrier by inducing accelerated maturation of ileal goblet cells and mucin-2 production. In addition, EGF treatment normalizes expression of crucial tight junction proteins in the ileum. Consequently, EGF treatment results in a significant decrease in intestinal paracellular permeability and improved barrier function. Results from these studies will provide significant contributions to the understanding of EGF-mediated reduction of NEC, which may lead to development of therapeutic strategies for the treatment of human NEC.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves.

No description available.

Cell-cell junctions are essential for tissue homeostasis. Prominent among these junctions are adherens junctions and tight junctions. Adherens junctions mediate adhesion between adjacent cells while tight junctions are responsible for establishing apical-basolateral polarity and limiting paracellular permeability. Loss or disruption of either adherens junctions or tight junctions leads to a myriad of disease states, thus these junctions need to be tightly regulated to prevent dysfunction. A unique property of tight junctions is their dependence on adherens junctions for proper assembly and maintenance. Loss or disruption of adherens junction leads to abnormal tight junctions. Understanding the mechanisms that mediate tight junction coupling to adherens junctions is important for treating diseases that arise from disrupted cell-cell junctions. Currently, two controversial models exist for how tight junctions are coupled to adherens junctions. In the first model, the adherens junction protein α-catenin is critical for tight junction assembly. The second model suggests that a second adherens junction protein, nectin is critical for tight junction assembly through binding the tight junction protein ZO-1, and disruption of tight junction assembly is independent of E-cadherin. α-catenin also binds ZO-1, but the consequences of this interaction are unknown. I hypothesized that α-catenin binding to ZO-1 plays a critical role in coupling tight junctions to adherens junctions. To test this, I mapped the ZO-1 binding site on α-catenin and engineered a point mutant of α-catenin that failed to bind ZO-1. Expression of this point mutant in epithelial cells showed that ZO-1 binding to α-catenin is essential for tight junction assembly and maintenance, while adherens junctions were unaffected. These findings established a role for ZO-1 binding to α-catenin in coupling tight junctions to adherens junctions during junction assembly, as well as at steady-state conditions. After discovering the importance of ZO-1 binding to α-catenin in coupling tight junctions to adherens junctions, I wanted to study whether this interaction is critical in a physiological setting. Tight junctions and adherens junctions are both strengthened in response to mechanical force; however the mechanisms responsible for tight junction strengthening were unknown. Using the system I previously developed, I show that ZO-1 binding to α-catenin is essential for increased tight junction integrity in response to mechanical force, coupling changes in tight junctions to increased stability of adherens junctions. Together, these findings identify a novel interaction that is critical for coupling tight junctions to adherens junctions under several conditions, and provide mechanistic insight into the cellular response to mechanical force.

published_or_final_version / Zoology / Master / Master of Philosophy

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Thesis (Ph.D. in Physiology and Biophysics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 89-98). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;