京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13158号 / 論医博第2145号 / 新制||医||1029(附属図書館) / (主査)教授 川上 浩司, 教授 戸井 雅和, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cells process a wide range of signals by means of multi-component receptors that span the plasma membrane. Our knowledge about the individual proteins involved in these signaling cascades has grown considerably over recent years. However, critical information about the detailed mechanisms of receptor activation, and the quantitative relationships between stimulus and biological response, is still missing. Here, I used the RET receptor tyrosine kinase (RTK), together with its glycosylphosphatidylinositol-coupled co-receptor GFRα3 and their activating growth factor artemin (ART), as a model system to investigate the quantitative and mechanistic features of receptor activation and signaling. I used a set of anti-RET agonist antibodies to induce different extents of receptor clustering on the cell surface, and studied how this factor affects the amplitude and kinetics of membrane-proximal and downstream signaling events, as well as the biological response of neurite outgrowth. Using simulations of the RET-GFRα3-ART system, I studied the effect of co-receptor involvement in the activation mechanism, as well as the importance of the specific activation pathway for the RET system’s response to variations in the expression levels of different components. The principal findings of my work include the following: 1) Higher order receptor clustering is required for full RET activation, as well as for the biological response of neurite outgrowth. 2) The activated forms of the receptor brought about by the agonist antibodies and by ART plus GFRα3 are identical with respect to the ability to activate the transient extracellular signal-regulated kinase (ERK) and Akt responses, but the antibodies show a reduced ability to induce sustained activation of ERK, Akt or c-Jun N-terminal kinase (JNK). 3) The involvement of GFRα3 co-receptor in the activation mechanism of RET provides cells with the ability to regulate their sensitivity to ligand without affecting the maximum amplitude of the pRET response. 4) This effect is limited if the co-receptor GFRα3 is pre-dimerized. Overall, my work aims to elucidate broad principles that underlie the quantitative relationships between RET activation, signaling, and the resulting cellular functional response, that can be applied to other receptor systems.

Development of tyrosine kinase inhibitors (TKIs) against oncogenic drivers has significantly improved survival of patients with oncogene-mutated non-small cell lung cancer (NSCLC). However, acquired resistance to TKIs emerges over time in essentially all patients who initially respond. Recent evidence suggests that drug-tolerant persister (DTP) cells, which survive and adapt to targeted therapies during an early phase of treatment, play an important role in the emergence of drug resistance. A previous study reported that cluster of differentiation 74 (CD74) expression is upregulated in epidermal growth factor receptor (EGFR)-mutated lung cancer after treatment with EGFR-TKIs and that CD74 can be one of the DTP cell markers. However, both the mechanism underlying CD74 expression and the role of CD74 in DTP cells remain unclear. In the current study, an attempt was made to identify the mechanism using cell culture systems and transgenic mouse models. The results confirmed CD74 upregulation at the messenger RNA (mRNA) level after treatments with TKIs in various oncogene-mutated cell lines, including those with EGFR mutations, ROS1 fusions, and ALK fusions. The class II transactivator (CIITA), upstream of CD74, and tumor necrosis factor (TNF)-α expression were induced by treatments with TKIs in tumor cells, leading to an increase in CD74 expression. In addition, the results showed that treatments with TKIs enhance the autocrine secretion of macrophage migration inhibitory factor (MIF), a ligand of CD74, from tumor cells. This implied that autocrine stimulation of CD74 signaling blocks apoptosis and causes emergence of DTP cells. To examine whether CD74 plays an important role in the emergence of resistance to TKIs in vivo, experiments were completed in which lung-specific EGFR-L858R-T790M transgenic mice were crossed with Cd74 knockout mice. The results showed that complete deletion of CD74 overcomes or delays resistance to TKIs. Taken together, the results of this study suggest that the MIF-CD74 axis can be a novel target to overcome resistance in driver-mutated NSCLC. / 2026-02-28T00:00:00Z

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Le neutrophile humain est impliqué dans la première ligne de défense du corps humain. Pour jouer ce rôle le neutrophile doit se rendre au site inflammatoire et phagocyter les agents microbiens. L'objectif de cette étude est d'étudier les mécanismes impliqués lors de la chimiotaxie des neutrophiles et la phagocytose. Les résultats obtenus permettent d'identifier plusieurs voies de signalisation impliquées dans ces deux réponses majeures du neutrophile et décrivent des différences majeures entre les signaux chimiotactiques qui permettent une spécialisation de la réponse.

Traumatic brain injury (TBI) can cause a number of deleterious effects to the neurovascular system, including reduced cerebral blood flow (CBF), vascular regression, and ischemia, resulting in cognitive decline. Research into therapeutic targets to restore neurovascular function following injury has identified endothelial EphA4 receptor tyrosine kinase as a major regulator of vascular regrowth. The research outlined herein utilizes an endothelial-specific EphA4 knockout mouse model (KO-EphA4flf/Tie2-Cre) to determine the extent to which this receptor may influence vascular regrowth following TBI. Analysis of the colocalization and proximity of endothelial and mural cell markers (i.e. PECAM-1 and PDGFRβ, respectively) in immunohistochemically-stained brain sections demonstrates that EphA4 silencing does not seem to affect the physical association between, nor total amounts of, endothelial cells and pericytes, between genotypes by 4 days post-injury (dpi). Nevertheless, these measures demonstrate that these cell types may preferentially proliferate and/or expand into peri-lesion tissue in both KO-EphA4flf/Tie2-Cre) and WT-EphA4fl/fl mice. These data further suggest that both genotypes experience homogeneity of PECAM-1 and PDGFRβ expression between regions of the injury cavity. Gene expression analysis using mRNA samples from both genotypes reveals that KO-EphA4flf/Tie2-Cre CCI-injured mice experience increased expression of Vegfa, Flt1, and Fn (Fibronectin) compared to sham-injured condition knockouts. These results demonstrate changes in expression of angiogenic factors in the absence of early differences in patterns of vessel formation, which may underlie improved vascular regrowth, as well as outline a potential mechanism wherein the interplay between these factors and EphA4 silencing may lead to improved cognitive outcomes following TBI. / Master of Science / Every day in the United States, an average of 155 people die due to the consequences of traumatic brain injury (TBI), with many survivors suffering life-long debilitating effects, including deficits in behavior, mobility, and cognitive ability. Because of this, there is a need for researchers to identify therapeutic strategies to stimulate recovery and improve patient outcomes. Recent advancements in the field of vascular biology have identified the regrowth of the blood vessels in the brain following TBI-induced damage as an important step in the recovery process, since the resulting increases in blood flow to damaged tissue will provide oxygen and nutrients necessary to fuel recovery. The work presented in this Masters thesis follows in this vein by examining a protein receptor known as EphA4, which is found on cells within blood vessels and has been implicated in reducing the rate of vessel growth under injury conditions. By blocking the activity of EphA4, we hoped to find increased vascular regrowth following brain injury in mice. During the experiments outlined herein, it was found that there were no statistically significant differences in vessel-associated cell densities between mice with or without EphA4 activity 4 days after injury, but there were differences in the levels of proteins and/or signals associated with vessel growth. Based on these results, we conclude that removing EphA4 activity increases expression of these pro-vessel growth proteins in mouse brains following injury at these early time points, potentially leading to increased vessel growth and improved recovery over subsequent weeks following injury.