Epithelial cell polarisation is a prerequisite for the branching morphogenesis in several organs. Differential screening techniques were used to identify genes, which are upregulated during induction of epithelium in early kidney development. This investigation revealed two separate genes, Nuclear localising protein 1 (Nulp1), a previously undescribed gene with sequence characteristics of the basic helix-loop-helix transcription factor family, and glycoprotein 210 (gp210, POM210), an integral membrane protein constituent of the nuclear pore complex (NPC). Of these, gp210 was found to be upreglated during conversion of mesenchyme to epithelium. The nuclear envelope, which demarcates the nuclear region in the eukaryotic cell, consists of an inner and an outer membrane that are fused at the locations for NPCs. These large macromolecular assemblages are tube like structures connecting the cytoplasmic and nuclear compartments of the cell. NPCs serve as the only conduits for exchange of molecular information between these cellular rooms. Electron microscopy techniques have revealed detailed information about the NPC architecture. A number of proteins (nucleoporins) have been characterised and embodied as components of the NPC structure. Active, energy dependent nucleocytoplasmic transport of RNAs and proteins is mediated by a group of soluble receptor proteins, collectively termed karyopherins. Gp210 has been suggested to be important for nuclear pore formation. Nevertheless, our analyses showed a limited expression pattern of gp210, with its mRNA and protein largely confined to epithelial cells in the mouse embryo. Furthermore, in several cell lines, gp210 was undetectable. The expression pattern of gp210 was not synchronised with some other nucleoporins, indicating NPC heterogeneity. Characterisation of the structure of the human gp210 gene, including its promoter region, gave insight about possible cell-type specific gene regulatory mechanisms. Regulation of molecular traffic between the nucleus and the cytoplasm leads to transcriptional control. Cell specific configuration of the NPC structure, due to diffential expression of gp210, could be involved in this control. Gp210 could be of importance for the development of epithelial cell polarisation.

<p>The urinary tract is one of the most intractable mucosal surfaces for pathogens to colonize. In addition to the natural barriers at this site, potential pathogens have to contend with the vigorous local innate immune response that is initiated by engagement of surveillance molecule TLRs. TLR4 appears to be not only exclusively expressed on superficial BECs but also critical to triggering robust local innate immune responses. TLR4 recognizes Gram-negative bacterial component LPS and initiates a series of intracellular NF-kappaB associated signaling events resulting in a cytokine response. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by E. coli and isolated LPS, and observed that, in addition to the classical NF-kappaB associated pathway, BEC TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, AC3 generated cAMP, and the transcriptional factor CREB. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract. </p><p>Here, we also report two additional distinct TLR4-mediated defense mechanisms in BECs. First, BEC TLR4 inhibits bacterial invasion, a necessary step for successful infection. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Additionally, we found that BECs continue to fight UPEC even after bacterial invasion by triggering bacterial exocytosis through a distinct TLR4-mediated mechanism following activation by LPS. In addition, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. The ability of TLR4 to mediate the rapid cytokine response, the inhibition of bacterial invasion, and the expulsion of intracellular bacteria from infected cells represents three previously unrecognized functions for this innate immune receptor.</p> / Dissertation

Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker of the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone, and that epiboly is inhibited when embryos are treated with Withaferin-A, a vimentin-targeting drug. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane, is notably absent from the blastoderm edge, and shows three distinct morphological regions approaching the leading edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, β-catenin, and E-cadherin, were present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, and not proliferation. Taken together, our data suggest that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and could serve as an in-vivo model for cancer and wound healing studies.

The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.

In response to extra-cellular cues, cells activate signal transduction pathways to elicit a biological response. Cell surface growth factor receptors such as the Met receptor tyrosine kinase (RTK) activate signals that result in cellular proliferation, survival, migration, as well as epithelial morphogenesis. In order for signal transduction to occur, docking proteins are recruited to the activated RTK, become phosphorylated on tyrosine residues, which then serve as docking sites for the recruitment of other signaling proteins. Docking proteins function to diversify the signal by assembling multi-protein complexes. The Gab1 docking protein is the most tyrosine phosphorylated protein upon Met receptor activation and is required for Met mediated signaling and biology. / Gab1 belongs to a family of docking proteins including the highly related Gab2 protein. Gab1 promotes signals for epithelial morphogenesis downstream of the Met receptor, however Gab2 is unable to do so. Insertion of the Gab1 Met binding Motif (MBM) which confers direct binding to the Met receptor, as well as membrane targeting of Gab2 is sufficient to switch the capacity of Gab2 to activate the morphogenic program, cell scatter and lamellipodia formation. This is achieved via activation of sustained signaling pathways, and redistribution of the Gab protein, and associated molecules to sites of lamellipodia formation at the peripheral edge of the cell. / Activation of the Met RTK, promotes the formation of dorsal ruffles on the apical surface of epithelial cells. The Met receptor, Gab1 and Gab1 associated molecules Shp2, Crk, and p8S subunit of PI3K, are localized to these structures, however only the Gab1erk complex is required to drive dorsal ruffle formation. Gab1 is required for Met induced dorsal ruffles as well as downstream the PDGF and EGF RTKs. These are a signaling micro-environment which results in enhanced receptor degradation. Inhibition or enhancement of Met mediated dorsal ruffle formation correlates with receptor stability. / Dorsal ruffle formation downstream of Met requires the enzymatic activity of PI3K and PLCgamma, both enzymes that metabolize PIP2, and form complexes with Gab1 downstream of Met. PLCgamma and the PIP3 lipid product of PI3K are co-localized with Gab1 in dorsal ruffles. Gab1 engages with elements of the cytoskeleton, actin and cortactin, providing a link between growth factor signaling and remodeling of the actin cytoskeleton. Gab1 is localized to membrane protrusions of the basal surface in organoid cultures and is required for actin protrusions of the basal surface of breast cancer cells.

The increase in ASMC mass is a major structural change described in airway remodeling in asthma. This increase has been attributed to ASMC hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced suggesting expansion of the muscle bundle towards the epithelium. Recent studies have suggested a role of epithelial derived chemokines in ASMC migration toward the epithelium. We hypothesized that chemokines (Eotaxin, RANTES, MIP-1alpha and IL-8) can directly influence ASMC mass by increasing the rate of proliferation or enhancing survival. ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8 or MIP-1alpha. To test for proliferation, stimulated ASMC were pulsed with 3H-thymidine or stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V and flow cytometry. Expression of phosphorylated p42/p44 and MAPKinases was assessed by Western analysis. In a concentration-dependent manner, chemokines such as Eotaxin, RANTES, IL-8 and MIP-lalpha increased ASMCs 3H-thymidine incorporation and DNA synthesis. Eotaxin, RANTES and IL-8 decreased the number of apoptotic ASMCs compared to the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. We conclude that chemokines might contribute to airway remodeling by increasing the number of ASMCs.

Previous studies have provided evidence that endothelial cell-based potassium ion selective electrodes possess the ability to quantify substances that have permeability-altering effects on those endothelial cells. The capability of these so-called biosensors to detect elevated concentrations of certain chemical agents found following tumor formation make them useful in the application as an alternative tumor angiogenesis assay. In this study an epithelial cell line, human colon adenocarcinoma epithelial cells (Caco-2), was used to fabricate membranes that were used to test concentrations of these chemical agents, known as cytokines, mimicking the concentrations that have been observed in the serum of healthy individuals as well as the higher concentration found in individuals with cancer. Additionally background information is provided related to the development of whole cell-based biosensors, metabolic pathways related to tumor angiogenesis and the subsequent increase in cytokine concentration, properties of the Caco-2 cell line that make them useful for the application in cell-based biosensors, and the ultimate effect the cytokines have on the permeability of the cells.

Die Kultivierung equiner Trachealepithelzellen stellt ein nützliches Modell dar, die (patho)-physiologischen Mechanismen der obstruktiven Atemwegserkrankungen des Pferdes auf zellulärer Ebene zu untersuchen. Ziel dieser Arbeit war es, Methoden für die Isolation, Charakterisierung und weitergehende Kultivierung equiner Trachealepithelzellen (ETEZ) zu etablieren und validieren und die Expression und Funktionalität der beta-adrenergen Rezeptoren an frisch isolierten ETEZ und deren Primärkulturen mittels pharmakologischer und biochemischer Verfahrenstechniken zu analysieren. Epithelzellen wurden durch Trypsinverdau aus der Trachea gesunder Pferde gewonnen, indem zuerst die Mukosa der Trachea freigelegt und diese dann vom daruntergelegenen Bindegewebe stumpf getrennt wurde. Das gewonnene Gewebe wurde zerkleinert und enzymatisch mit 0,25% Trypsin-EDTA-Lösung für 2 h bei 37°C verdaut. Durch Siebung und Zentrifugation wurden die Zellen gereinigt, vereinzelt und gesammelt, wobei kontaminierende Fibroblasten später durch differentielle Adhäsion von den Epithelzellen getrennt wurden. Die isolierten Zellen wurden sowohl licht- bzw. elektronenmikroskopisch charakterisiert, als auch immunzytochemisch hinsichtlich Zytokeratin (für Epithelzellen) und Vimentin (für Fibroblasten) gefärbt. Die durchschnittliche Zellausbeute wurde mit der Neubauer-Zählkammer bestimmt und betrug 6,10 ± 0,63×106 Zellen pro 500 mg zerkleinertem Gewebe (n = 11). Die Zellvitalität wurde mittels Trypanblau-Färbung ermittelt und betrug 94,70 ± 1,17% (n = 11). Immunfluoreszensfärbungen zeigten, dass 93,57 ± 1,67% (n = 11) der frisch isolierten Zellen und ca. 100% (n = 5) der Primärkulturen auf Zytokeratin 5/6/18 positiv reagierten. Auf Anti-Vimentin reagierten dagegen nur 9,83 ± 0,94% (n = 11) der Zellen positiv. Die Zellen wurden in einer Dichte von 6,90 x 104 Zellen/cm2 in serumfreiem AECGM ausgesät und bildeten innerhalb einer Woche einen konfluenten Monolayer. Die konfluenten Zellen wurden mittels Dispase II abgelöst. Die erste (P1) und die zweite (P2) Passage konnte erfolgreich in serumfreien AECGM kultiviert und auf der Stufe P2 30 Tage lang gehalten werden. Weitethin wurden die Expression und Funktionalität der b-adrenergen Rezeptoren in frisch isolierten und kultivierten Epithelzellen untersucht. Mittels Radioligandenbindungsstudien, Westernblot, Immunfluoreszensfärbung und cAMP-Assays konnten erstmalig die Dichte, Affinität, Subtypen, Proteinexpression und zelluläre Lokalisation der beta-adrenergen Rezeptoren sowie die Rezeptorfunktion bestimmt werden. Messungen an frisch isolierten ETEZ ergaben für die mittlere Dissoziationskonstante (KD) von 31,78 ± 6,57 pM (n = 7) und eine maximale b-adrenerge Rezeptordichte (BMax) von 12727 ± 883,6 Bindungsstellen/Zelle (n = 7) ermittelt aus Sättigungsexperimenten mit dem b-adrenergen Rezeptorantagonisten [125I] Iodocyanopindolol (ICYP) in Anwesenheit des nicht selektiven beta-Rezeptorantagonisten (±)-CGP 12177. Für Primärkulturen ergaben sich Werte für KD von 15,26 ± 3,37 pM (n =6) und für BMax von 3730 ± 212 Bindungsstellen/Zelle (n = 6). Bei Verdrängungsexperimenten wurde die ICYP konzentrationsabhängig durch den beta2-selektiven Rezeptorantagonisten ICI 118.551 und den beta1-selektiven Rezeptorantagonisten CGP 20712A verdrängt, wobei für ICI 118.551 eine 10.000-fach höhere Affinität (Ki = 1,74 ± 0,15 nM in frisch-isolierten Zellen und 1,19 ± 0,41 nM in Primärkultur) gezeigt wurde als für CGP 20712A (Ki = 17 ± 7,90 μM in frisch isolierten Zellen). Die cAMP-Bildung wurde in frisch isolierten ETEZ konzentrationsabhängig durch die beta-adrenergen Rezeptoragonisten Isoproterenol, Epinephrin und Norepinephrin in der Reihenfolge ihrer Potenz mit einer EC50 von 58 nM (n = 6), 13,60 μM (n = 6) bzw. 0,43 mM (n = 6) stimuliert. Diese cAMP-Bildung konnte durch Behandlung der Zellen mit 100 nM der beta2-selektiven ICI 118.551, nicht aber durch 300 nM des beta1-selektiven CGP 20712A blockiert werden. Mit einem beta2-adrenergen Rezeptorantikörper konnte eine 72 kDa Proteinbande und mit demselben Antikörper in der Fluoreszenzfärbung Rezeptorantigene auf der Zelloberfläche nachgewiesen werden. Zusammenfassend konnten mit dem etablierten Protokoll große Mengen equiner Trachealepithelzellen isoliert und kultiviert werden. Die Ergebnisse dieser Studie zeigen erstmalig, dass primäre equine Trachealepithelzellen funktionale beta2-adrenerge Rezeptoren exprimieren und das Protokoll zur Etablierung eines zellbasierten Modells geeignet ist, um in vitro verschiedene Funktionen und eine Pharmaka-induzierte Regulation der beta-adrenergen Signalkaskade hinsichtlich physiologischer und pathophysiologischer Zustände bei Atemwegserkrankungen des Pferdes und hierfür relevante pharmakologische und toxikologische Zielstrukturen untersuchen zu können.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.