The integrin α6β4 (referred to as β4) is expressed in epithelial cells where it functions as a laminin receptor. Integrin β4 is important for the organization and maintenance of epithelial architecture in normal cells. Particularly, β4 is shown to be essential for mammary gland development during embryogenesis. Integrin β4 also plays important roles in tumor formation, invasion and metastasis in breast cancer. However, the mechanism of how integrin β4 mediates breast tumor formation has not been settled. A few studies suggest that integrin β4 is involved in cancer stem cells (CSCs), but the mechanism is not clear. To address this problem, I examined the expression of β4 in breast tumors and its potential role involved in regulating CSCs. My data shows that β4 is expressed heterogeneously in breast cancer, and it is not directly expressed in CSCs but associated with a basal epithelial population. This work suggests that β4 can regulate CSCs in a non-cell-autonomous manner through the interactions between β4+ non-CSC population and β4- CSC population. My data also shows that β4 expression is associated with CD24+CD44+ population in breast tumor. To further study the role of β4 in breast cancer progression, I generated a β4 reporter mouse by inserting a p2A-mCherry cassette before ITGB4 stop codon. This reporter mouse can be crossed with breast tumor models to track β4+ population during tumor progression.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13195号 / 論医博第2159号 / 新制||医||1030(附属図書館) / (主査)教授 松田 秀一, 教授 三森 経世, 教授 妻木 範行 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Microsporidia are a distant fungal pathogen that have severe clinical consequences for the immunocompromised. Previous work identified a microsporidian pathogen protein termed Microsporidian ADAM or MADAM. This protein has close sequence homology to other ADAM proteins (A Disintegrin and Metalloproteinase) in two microsporidian species, Encephalitozoon intestinalis and E. cuniculi. ADAM proteins have a wide range of functions, including binding to integrins and host signaling. It is known that many pathogens manipulate integrins to invade host cells, and it is predicted that microsporidia are also exploiting this host target. Previous work with the MADAM protein demonstrated that this protein has a role in adherence to host cells. Separate work showed integrin inhibitors can also decrease spore adherence to cells. Experiments in this project complement previous research and further characterize the binding of microsporidia to host integrins and the intracellular consequences of that binding. This work found the integrin binding sequence of MADAM (MADAM peptide) is important for spore binding to host cells. Separate work shows that the host β1 integrin is also involved in spore adherence. Additional work demonstrated that spores and the MADAM peptide elicited an increase in host integrin signaling in Western blotting experiments. And finally, preliminary acellular interferometry experiments suggest the MADAM protein binds specifically to α5β1 and α6β4 integrins. Together, these results suggest microsporidia spores rely, in part, on host integrins to bind to host cells before infection.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24476号 / 医博第4918号 / 新制||医||1062(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 小濱 和貴, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

Vascular endothelial growth factor (VEGF) is required for the development, growth and survival of blood vessels. Endothelial cell behavior is altered by cell substrate stiffness, suggesting that VEGF activity might also be influenced by cell-substrate mechanics. We studied VEGF binding, internalization, and signaling as a function of substrate stiffness using endothelial cells cultured on fibronectin (fn) linked polyacrylamide gels. Individual cell analysis of VEGF-induced calcium fluxes in endothelial cells on various stiffness extracellular matrices (ECM) revealed heterogeneity in our cell population that would have been lost using population based averaging. Cluster analysis of individual cells identified two key groups of reacting cells- a minor fraction of highly reactive cells and the bulk of the cells with minimal activation. At subsaturating VEGF doses, highly active cells were phenotypically smaller and thinner than the bulk population. Overall, cells on our softest substrates (4 kPa) were most sensitive to VEGF. To better understand the mechanisms underlying the changes in VEGF signaling due to stiffness, we explored how matrix binding of VEGF and tethering of cells to the matrix modulates VEGF processing. VEGF-ECM binding was enhanced with heparin pre-treatment, which exposed a cryptic VEGF binding site in the fn ECM. Cell produced ECM on the softest substrates were least responsive to heparin, but the cells internalized more VEGF and showed enhanced VEGF signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF in all conditions. β1 integrin, which connects cells to fn, modulated VEGF uptake in a stiffness dependent fashion. β1 protein levels were consistent with stiffness, yet cells on hard surfaces showed greater decreases in VEGF internalization than cells on softer matrices after β1 inhibition. Stiff matrices facilitate the unfolding of fn, which may reduce the binding capacity of β1 integrin. Thus a greater proportion of activated β1 integrin may be sensitive to inhibition in the stiff condition as compared to the soft. Ultimately, through analysis of individual and population-based VEGF-cell responses to stiffness, this study provides insight into how signaling dynamics, cell heterogeneity, and microenvironment influence tissue regeneration and response to injury and disease.

Improving the therapeutic efficacy and quality of life of patients by reducing the side effects caused by non-specificity of cytotoxic drugs has been a challenge in cancer treatment. A hypothesis was developed where integrin binding induced conformational change in a drug conjugated to hairpin peptide with an integrin binding ligand can lead to preferential accumulation of drug and reduced collateral damage by decreased premature prodrug activation. A model drug, MTX and a tripeptide ligand, RGD, known to specifically bind tumor overexpressing α v β 3 integrin receptors, were selected to test the hypothesis. A twelve amino acid sequence that has been previously shown to preferentially adopt an anti-parallel beta hairpin conformation in aqueous environment was flanked with MTX and RGD on N and C termini respectively by solid phase peptide synthesis to form a labile link between Arg-Glu specifically cleaved by SGPE, a Streptomyces griseus derived endopeptidase. Adenoviral vector was developed using AdEasy system for β 3 cDNA transfection to overexpress integrin α v β 3 receptor. MTX-α-RGD and MTX-β-hairpin-RGD were characterized using MALDI-TOF (MTX-α-RGD, 782.6(M+H + ); MTX-β-hairpin-RGD, 2272.1(M+H + )). Cell adhesion assay using HUVEC and A549 cells that overexpress α v β 3 showed that RGD conjugated prodrugs recognize and preferentially bind to integrin α v β 3 in RGD dependent manner. In rabbit plasma, MTX-β-hairpin-RGD was found to be 3 times more stable than MTX-α-RGD. In the absence of α v β 3 binding, SGPE mediated hydrolysis rate of MTX-β-hairpin-RGD was 0.7±0.1 ng/hr, that was significantly (P<0.025) lower than that of MTX-α-RGD (1.0±0.1ng/hr), a prodrug without hairpin structure. In presence of α v β 3 over-expressing cells, significant increase (P<0.025) in hydrolysis rate of MTX-β-hairpin-RGD to 1.0±0.1 ng/hr was observed, not significantly (P=0.6) different from that of MTX-α-RGD (1.1±0.1ng/hr). In addition, there was 400% increase in the fluorescence when FRET based quenching was abolished by the binding induced unfolding. These experiments along with docking studies using molecular modeling support the binding induced unfolding. Results from this investigation suggest that drugs conjugated to peptide ligands such as RGD may reduce the dose needed to achieve therapeutic concentrations by preferential recognition and binding to overexpressed integrin markers. Secondly, reduction of premature activation of prodrugs and thus reduced collateral damage may be achieved by making the the drug release to occurs preferentially upon binding to cells expressing specific integrin markers.

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals (including us humans) and Fungi. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. Here we have isolated a naked eukaryotic amoeba with filose subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a wholistic approach including light, electron, fluorescence microscopy and SSU rDNA, we find that this amoeboid organism fails to match any previously described eukaryote genus. Our isolate amoebae are most similar to some variosean amoebae which also possess acutely pointed filose subpseudopodia. Maximum likelihood and Bayesian tree of the SSU-rDNA gene places our isolate in Variosea of Amoebozoa as a novel lineage with high statistical support closely related to the highly diverse protosteloid amoebae Protostelium. This novel variosean is herein named “Hodorica filosa” n. g. n. sp. We present a robust phylogeny of Amoebozoa based on a broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea and Tevosa. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features Integrins are transmembrane receptors that activate signal transduction pathways upon extracellular matrix binding. The Integrin Mediated Adhesion Complex (IMAC), mediates various cell physiological processes and are key elements that are associated animal multicellularity. The IMAC was thought to be specific to animals. Over the last decade however, the IMAC complexes were discovered throughout Obazoa. We show the presence of an ancestral complex of integrin adhesion proteins that predate the evolution of the Amoebozoa. Co-option of an ancient protein complex was key to the emergence of animal multicellularity. The role of the IMAC in a unicellular context is unknown but must also play a critical role for at least some unicellular organisms.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13502号 / 論医博第2261号 / 新制||医||1061(附属図書館) / (主査)教授 村川 泰裕, 教授 柳田 素子, 教授 長船 健二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM