Receptor interacting protein kinase 1 (RIPK1) is an enzyme acting downstream of tumor necrosis factor alpha to control cell survival and death. RIPK1 expression has been reported to cause drug resistance in cancer cells; but so far, no published studies have investigated the role of RIPK1 in vitamin D action. In the present study, we investigated whether RIPK1 played any role in 1,25-dihydroxyvitamin D3 (1,25D3)-induced growth suppression. In our studies, RIPK1 decreased the transcriptional activity of vitamin D receptor (VDR) in luciferase reporter assays independently of its kinase activity, suggesting a negative role of RIPK1 in 1,25D3 action. RIPK1 also formed a complex with VDR and deletion analyses mapped the RIPK1 binding region to the C-terminal ligand-binding domain of VDR. Subcellular fractionation analyses indicated that RIPK1 increased VDR retention in the cytoplasm, which may account for the inhibition of VDR transcriptional activity. Consistent with the reporter analyses, 1,25D3-induced growth suppression was more pronounced in RIPK1-null mouse embryonic fibroblasts (MEF) and RIPK1 knockdown ovarian cancer cells than control cells. We have also shown that VDR was involved in RIPK1-mediated cell death pathway in a cell line specific manner. In vivo study showed that VDR deletion delayed the necroptotic response to tumor necrosis factor alpha in mice. Western blot analyses of platinum sensitive and resistant cell lines showed a correlation between RIPK1 expression and drug resistance, suggesting a possible role of RIPK1 in drug resistance. In conclusion, this study is the first to define RIPK1 as a VDR repressor, projecting RIPK1 depletion as a potential strategy to increase the potency of 1,25D3 and its analogs for cancer intervention.

Cell death is an imperative mechanism for the development, homeostasis and survival of an organism. Various forms of cell death have been documented and recent reports indicate that the mode of cell death elicited can have a profound influence on the development and perpetuation of inflammation. Apoptosis is the predominant, programmed pathway of cell death, which ensures physiological elimination of unwanted cells. On the other hand, another cell death pathway described as programmed necrosis (necroptosis), has recently been revealed. The induction of necroptosis and its impact in host biology is not clear. Herein I have evaluated the mechanisms of necroptosis in macrophages, an important cell type of the immune system. My experiments indicate that type I interferon (IFN-I) signaling through transcription factors STAT1, STAT2 and IRF9, collectively described as the ISGF3 complex, is indispensable for necroptosis of macrophages. Furthermore, my results indicate that IFN-I signaling promotes the sustained phosphorylation of receptor interacting protein kinase 3 (Rip3), a key protein required for the execution of necroptosis. My findings also reveal that dynamin-dependent endocytosis following IFNβ stimulation and caspase inhibition is necessary for the induction of necroptosis. The results presented in this thesis provide new insights into the molecular mechanisms of necroptosis and therefore contribute to a deeper understanding of multiple inflammatory pathologies.

Cell death plays a vital role throughout the immune response, from the onset of inflammation to the elimination of primed T cells. Understanding the regulation of cell death within immune cells is of vital importance to understanding the immune system and developing therapies against various immune-disorders. In this thesis I have investigated the regulation of cell death and its functional role in of the innate and adaptive arms of the immune system. The mechanisms that govern expansion and contraction of antigen stimulated CD8+ T cells are not well understood. In the first section of this thesis, I show that caspase-3 becomes activated in proliferating CD8+ proliferation, yet this does not result in cell death. I used both in vivo and in vitro models to demonstrate that caspase-3 activation is specifically driven by antigen presentation and not inflammation, and that it likely plays a role in promoting T cell proliferation. Next, I present novel data regarding the regulation of a newly identified form of programmed cell death via necrosis, known as necroptosis. I show that the cellular inhibitor of apoptosis (cIAP) proteins act to limit activation of key necroptosis proteins in macrophage cells. Furthermore, I show that necroptosis can be exploited by intracellular bacterial pathogens to escape removal by the immune system. I also demonstrate that necroptosis is highly intertwined with the pathway of inflammation, and the autocrine production of type-I interferon constitutes a vital positive feedback loop in the induction of inflammatory cell death. In the final section of my thesis work, I delve into the specific regulation of Rip1 kinase and demonstrate that in addition to previously demonstrated regulation by caspase-8, cathepsins are also able to cleave Rip1 kinase and limit necroptosis. This thesis presents a wide variety of novel data regarding the regulation of cell death within immune cells. In total, the results reveal a picture of two divergent forms of programmed cell death, apoptosis and necroptosis. Through improving the understanding of the cross-regulation of these two key cell death pathways this work aims to improve the understanding of the immune function.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13281号 / 論生博第19号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Since several different pathways are involved in cerebral ischemia/reperfusion injury, combination therapy rather than monotherapy may be required for efficient neuroprotection. In this study, we examined the protective effects of an apoptosis inhibitor Gly 14-humanin (HNG) and a necroptosis inhibitor necrostatin-1 (Nec-1) on hypoxia/ischemia/reperfusion injury. Cultured mouse primary cortical neurons were incubated with Nec-1, HNG or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay at 24 h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4 h after reperfusion. Neurological deficits were evaluated and the cerebral infarct volume was determined by TTC staining. Nec-1 or HNG alone had protective effects on OGD-induced cell death. Combined treatment with Nec-1 and HNG resulted in more neuroprotection than Nec-1 or HNG alone. Treatment with HNG or Nec-1 reduced cerebral infarct volume from 59.3 ± 2.6% to 47.0 ± 2.3% and 47.1 ± 1.5%, respectively. Combined treatment with HNG and Nec-1 improved neurological scores and decreased infarct volume to 38.6 ± 1.5%. In summary, we demonstrated that the combination treatment of HNG and Nec-1 conferred synergistic neuroprotection on hypoxia/ischemia/reperfusion injury in vitro and in vivo. These findings provide a novel therapeutic strategy for the treatment of stroke by combining anti-apoptosis and anti-necroptosis therapy.

No description available.

Yes / The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone. / Funded in part through a grant to RA from the Cultural Bureau of the Kingdom of Saudi Arabia.

No description available.

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Influenza A viruses (IAV) are negative sense RNA viruses that naturally infect a wide variety of animals. Different subtypes of the virus infect waterfowl, poultry, pigs, horses, ferrets, bats, dogs, cats and humans, causing zoonotic outbreaks and pandemics. In humans, IAV strains cause seasonal epidemics that can result in up to 50000 deaths and 700000 hospitalizations each year, and ranks in the top ten causes of death in the United States. In addition, virulent strains of IAV have caused pandemic outbreaks triggering numerous fatalities. While tropism to the upper respiratory tract is important for virus transmission, infection of the lower lung is most correlated with pathogenesis. The mammalian lung has multiple structural cell types, of which two classes are considered most important for IAV pathogenesis. These are (1) alveolar unit cells and (2) cells of the conducting airways, primarily those of the bronchi and bronchioles. Alveolar unit cells, or pneumocytes, chiefly comprise type I and type II alveolar epithelial cells, and are involved in gas exchange and surfactant production. Cells of the bronchi and bronchioles (e.g., basal, secretory, ciliated, club, goblet and neuroendocrine cells) perform numerous functions related to tissue repair/renewal, and mucous production. Upon IAV infection the regulated (or programmed) death of the infected cell represents an important pathogen clearance mechanism. Programmed cell death can be largely non-inflammatory (e.g., apoptosis) or pro-inflammatory (e.g., necroptosis). In this dissertation, I outline experiments carried out to identify the role of pro-inflammatory programmed cell death in influenza A virus clearance and pathogenesis both in vitro and in vivo. My work outlines the role of necroptosis in IAV clearance, and how this controlled form of cell death, particularly in alveolar unit cells, can be exploited as a potential new therapeutic avenue for severe influenza disease. / Cancer Biology & Genetics