In several human cancers, ErbB2 over-expression facilitates the formation of constitutively active homodimers resistant to internalization which results in progressive signal amplification from the receptor, conducive to cell survival, proliferation, or metastasis. Here we report on studies of the influence of ErbB2 over-expression on localization and signaling in polarized Caco-2 and MDCK cells, two established models to study molecular trafficking. In these cells, ErbB2 is not over-expressed and shares basolateral localization with ErbB3. Over-expression of ErbB2 by transient transfection resulted in partial separation of the receptors by relocalization of ErbB2, but not ErbB3, to the apical surface, as shown by biotinylation of the apical or basolateral surfaces. These results were confirmed by immunofluorescence and confocal microscopy. Polarity controls indicated that the relocalization of ErbB2 is not the result of depolarization of the cells. Biotinylation and confocal microscopy also showed that apical, but not basolateral ErbB2 is activated at tyrosine 1139. This phosphotyrosine binds adaptor protein Grb2, as confirmed by immunoprecipitation. However, we found that it does not initiate the canonical Grb2-Ras-Raf-Erk pathway. Instead, our data supports the activation of a survival pathway via Bcl-2. The effects of ErbB2 over-expression were abrogated by the humanized anti-ErbB2 monoclonal antibody Herceptin added only from the apical side. The ability of apical ErbB2 to initiate an altered downstream cascade suggests that subcellular localization of the receptor plays an important role in regulating ErbB2 signaling in polarized epithelia.

Germ-free animals have been used to study the effects of microbiota for several decades. In that time, numbers of differences from specific-pathogen-free (SPF) animals have been reported, including differences in absolute numbers or percentages of various immune populations, enormously enlarged coecum and lack of germinal centers. However, many of the crucial information about structural and functional differences in their secondary lymphoid organs still remains uncovered. With novel microscopical approaches, such as light sheet fluorescent microscopy, enabling 3D visualization of whole samples without processing them to a series of slides, and multicolor cytometry, allowing the characterization of numbers of cellular populations within a matter of seconds and in a highly quantitative manner, the uncovering of fundamental differences finally seems to be within reach. MHC II-EGFP knock-in mouse model brings the advantages of a fluorescent protein expressed in physiological histological contexts into both fields. Lymphoid and other tissues can be visualized microscopically without the need of staining (even in vivo). Information about the expression of both plasma membrane-localized and intracellular MHC II in various tissues could be acquired directly. Combining MHC II-EGFP knock-in mouse model with...

The human colonic cell line T84, a model for studying epithelial chloride secretion and cystic fibrosis chloride channel (CFTR) function, was used to investigate the regulatory role of estrogens in transepithelial ion transport. Estrogens and other steroid hormones do not stimulate chloride secretion by themselves. However, 17 β-estradiol (17β-E2) rapidly (within seconds to minutes) potentiates carbachol- and thapsigargin-stimulated chloride secretion measured as short circuit current in voltage-clamped T84 monolayer cultures. The cholinergic agonist carbachol and the SR Ca2+ ATPase inhibitor thapsigargin stimulate chloride secretion by elevating intracellular calcium. 17α-estradiol, a stereoisomer that does not activate nuclear estrogen receptors, is equipotent with 17β-E2. Other non-estrogen steroids produce much less, if any, potentiation of calcium-stimulated chloride secretion. The estrogen receptor antagonist tamoxifen does not block 17β-E2 potentiation of calcium-stimulated chloride secretion, indicating that the classical estrogen receptors are not involved. Potentiation is greater when 17β-E2 is applied to the apical membrane than to the basolateral membrane. 17β-E2 effects on chloride secretion coincide with an increase in monolayer electrical conductance, which is consistent with activation of one or more ion channel species. Potentiation is not blocked by the chloride channel blockers DIDS and NPPB but is abolished by the PKA inhibitor H89, suggesting that 17β-E2 potentiation depends on the activity of CFTR but not other types of apical membrane chloride channels. 17β-E2 does not increase the activity of calcium-activated potassium channels in the basolateral membrane as measured in nystatin-permeabilized monolayers. 17β-E2 effects are not blocked by the MAP kinase kinase inhibitor PD 98059, or by the PKC inhibitor bisindoylmaleamide, suggesting that these signal transduction pathways are not involved. 17β-E2 potentiation requires extracellular Ca2+. Paradoxically, 17β-E2 reduces the rise in intracellular free Ca2+ levels in thapsigargin-stimulated T84 cells, as measured by fura-2 fluorescence. From my studies I conclude that 17β-E2 causes an increase in the sensitivity of T84 cells to calcium-elevating secretagogues. This effect may be due to nongenomic actions of 17β-E2 on CFTR function and/or the activity of store-operated calcium channels, which leads to a change in CFTR functional regulation.

Systemic steroid hormone and intracellular signaling pathways are known to act cooperatively during the development of vertebrate and invertebrate epithelia. However, the mechanism of this interaction is poorly understood. Morphogenesis of Drosophila leg imaginal disc epithelia is regulated both by the steroid hormone 20-hydroxyecdysone (ecdysone) and the RhoA GTPase signaling pathway. Recent evidence suggests that these pathways act cooperatively to control imaginal disc morphogenesis. Thus, leg imaginal disc morphogenesis is an excellent system in which to study the interaction of steroid hormone and intracellular signaling pathways. We have identified mutations in three genes, 12-5, 18-5, and 31-6, with roles in the morphogenesis of leg epithelia. Of particular interest, these mutations interact genetically with each other, mutations in the RhoA signaling pathway, and the ecdysone regulated Sb-sbd (Stubble) transmembrane serine protease. This suggests that the 12-5, 18-5, and 31-6 gene products may link hormone and RhoA signaling responses. The goal of this research was to identify and characterize the 18-5 and 12-5 genes in order to discern the mechanistic relationship between the RhoA pathway and ecdysone hierarchy.18-5 and 12-5 were precisely mapped to molecular locations within the Drosophila genome utilizing a P-element recombination mapping technique. This work narrowed the location of the 18-5 locus to within an interval of 112 kb within the Drosophila genome sequence. This interval contains 17 known and predicted genes. I also mapped the location of the 12-5 locus to a 2.6 Mb interval of the 2nd chromosome. Based on phenotypic analyses and the site of the molecularly mapped interval, a candidate gene for the 18-5 mutation was identified. Sequence analysis of the candidate gene was inconclusive and requires further analysis. Genetic interaction assays indicate that the 18-5 gene product acts upstream or at the level of Rho kinase in the RhoA signaling pathway.

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<p> Serotonin positive (5HT+) and tyrosine hydroxylase positive (TH+) cells were identified using fluorescent immunocytochemical methods and quantified in the gill epithelium of six species of fish. 5HT+ cells were located in the filament epithelium in contact with the basal lamina on the efferent side, and in the lamellar epithelium where they were occasionally exposed to the external milieu. Thus, these cells appear to represent two populations of neuroepithelial cells (NEC) as proposed in other studies. In trout, bass and killi fish, NECs were revealed exclusively in the primary epithelium. In tilapia, NECs were located exclusively in the secondary epithelium, whereas in perch and zebrafish they occurred in both epithelial layers. The two types of NECs varied in number both within and among the species. Seasonal comparisons of NECs in perch revealed a decrease in cell density in the filament between July and November, though there was no significant difference in the density of NECs in the lamellae. TH+ cells were identified in perch, zebrafish and killi fish. In zebrafish TH+ cells occurred in similar numbers to 5HT+ cells, and were generally present in similar locations. It is likely that in this case, many of the labelled cells were positive for both markers. In two of the species, perch and killi fish, the density and distribution was such that the TH+ cells and 5HT+ cells were unlikely to be the same. A quantitative comparison of total catecholamine (CA) stores, using high performance liquid chromatography (HPLC), revealed that gill tissues in general contained higher levels of epinephrine (EPI) than norepinephrine (NOR) and dopamine (DOP). Finally, attempts were made to determine whether NECs would survive in 2-4 day old cultures of dispersed gill cells from perch, using immunocytochemical labelling for 5HT. A few successful cases are presented.</p> / Thesis / Master of Science (MSc)

Thesis / Master of Science (MS)

Hippo signaling is a tumor suppressive signaling pathway that controls organ size by regulating cellular proliferation, apoptosis, and differentiation during development, regeneration, and homeostasis. The Hippo pathway inhibits transcriptional co-activators and Hippo pathway effectors YAP/TAZ, activation of which is often seen in cancer. Within the adult mammalian intestine, homeostasis of which requires intricate reciprocal interaction between the gut epithelium and adjacent mesenchyme, the Hippo-YAP pathway is crucial for intestinal epithelial homeostasis and regeneration. However, its role in adult mesenchymal homeostasis remains poorly understood. Here, I genetically dissect the role of mesenchymal Hippo-YAP signaling in adult intestinal homeostasis. I find that deletion of core kinases LATS1/2 or YAP activation in mesenchymal progenitor cells, but not terminally differentiated cells, disrupts signaling in the stem cell niche and mesenchymal homeostasis by inducing mesenchymal overgrowth and suppressing smooth muscle actin expression. Furthermore, inhibition of Hippo signaling in Gli1+ mesenchymal progenitors, the main source of Wnt ligands within the stem cell niche, stimulates Wnt ligand production and subsequent epithelial Wnt pathway activation, thereby driving epithelial regeneration following DSS-mediated injury as well as exacerbating APC-mediated tumorigenesis. Altogether, our data reveal a previously underappreciated requirement and the underlying mechanism for stromal Hippo-YAP signaling in adult intestinal homeostasis.