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염증해소 반응에 있어서 Myc-nick의 새로운 기능

By 종헌재


학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2018. 8. 서영준.Acute inflammatory response is characterized by sequential events including an immediate recruitment of polymorphonuclear leukocytes (PMNs), a subsequent clearance of leukocytes and an eventual resolution. Eventually, restoration of body homeostasis is accomplished when inflammation is resolved successfully, which is underpinned by adaptive immunity at the post-resolution phase. An impaired resolution causes chronic inflammatory response, leading to persistent tissue damage. In the past many years, researchers have focused on developing drugs such as non-steroidal anti-inflammatory drugs to treat patients with inflammatory diseases by shutting off inflammatory response. However, this is not effective in augmenting cellular adaptive response to acute inflammation through potentiation of innate immunity as they often interrupt the consecutive host-protective process. Recent work has been hence shifted to discovery of drugs that actively facilitate resolution of inflammation, achieving a termination of inflammatory response utilizing intrinsic mechanism and a simultaneous recovery of body homeostasis. To achieve the active resolution, two physiologic processes are employed. These include recruitment of leukocytes (typically neutrophils) that eventually die, mostly via apoptosis, after fighting infectious agents and subsequent clearance of dead cells and debris. Macrophages, especially pro-resolving macrophages, are typical cells that are effective on clearance of apoptotic cells by phagocytic engulfment (efferocytosis). These pro-resolving macrophages carries M2 (alternatively activated) phenotypic characteristics. <br/> Carcinogenesis is closely associated with chronic inflammation. Chronic inflammation can cause activation and accumulation of immunosuppressive cells, which favors initial tumor formation and later survival of tumor cells. In recent years, resolution of cancer-associated inflammation is becoming a new approach for cancer therapy. Pro-resolving treatment not only eliminates excessive inflammatory signal, but mediates a post-resolution reaction that generates adaptive immune response. The influx of adaptive immune cells possibly potentiate therapy as these cells have been demonstrated to be effective in immunotherapy. Therefore, it is of great interest to apply pro-resolving strategies for the treatment of inflammatory disorders. <br/> The transcription factor, c-Myc has been known to play a role in manifestation or maintenance of phenotypic characteristics of alternatively activated macrophages. Contradictory to this notion, I found that peritoneal macrophages from zymosan A-treated mice, in the M2-like state, exhibited a markedly reduced level of c-Myc. This was accompanied by accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, I noticed that the generation of Myc-nick promoted the M2 polarization of macrophages, enhanced the efferocytic capability of these cells and accelerated resolution of inflammation. Another study showed that c-Myc cleavage was not obviously affected by treatment of pro-resolving lipid mediator, resolvin D1 (RvD1), to HCT 116 cancer cells. This is probably due to a lack of Calpain activation during a pro-resolving treatment. It is notable that RvD1 exerts anti-cancer effect by facilitating c-Myc degradation in HCT 116 cells. My studies indicate that Myc-nick might be an innovative therapeutic target in macrophages but not cancer cells. Since tumor-associated macrophages (TAM) are largely present in tumor microenvironment, the role of Myc-nick in the function of TAM merits further investigation in the context of development of novel anticancer strategies. <br/>Chapter I 1<br/> 1.1. Introduction. 2<br/> 1.2. Resolution of inflammation. 3<br/> 1.3. Efferocytosis 8<br/> 1.4. Role of cytoskeleton during efferocytosis. 11<br/> 1.4.1. Actin cytoskeleton for efferocytosis 12<br/> 1.4.2. Microtubule cytoskeleton for efferocytosis 14<br/> 1.5. LC3-associated phagocytosis 15<br/> 1.6. Phenotypic characteristics of macrophage during resolution . 16<br/> 1.7. Role of c-Myc. 18<br/> 1.8. Myc-nick 19<br/> 1.9. Perspectives. 22<br/> <br/> Chapter II. 27<br/> 2.1. Abstract . 28<br/> 2.2. Introdutcion. 29<br/> 2.3. Materials and methods 32<br/> 2.3.1. Materials . 32<br/> 2.3.2. Methods. 33<br/> Cell culture . 33<br/> Zymosan- and thioglycollate-induced peritonitis 33<br/> Preparation of bone marrow derived macrophages (BMDMs) 34<br/> Isolation of mouse thymocytes and generation of apoptotic cells. 35<br/> Efferocytosis assay 35<br/> Flow cytometry assay . 36<br/> Immunofluorescent staining and imaging. 37<br/> Western blotting and PCR analysis. 38<br/> Bioinformatic Studies. 39<br/> Statistical analysis 40<br/> 2.4. Results 41<br/> 2.4.1. Murine macrophages have diverse characteristics at different stages 41<br/> 2.4.2. During the resolution of zymosan A-induced murine peritonitis, proinflammatory macrophages switch to pro-resolving macrophages with an M2-like phenotype 44<br/> 2.4.3. Full length c-Myc is cleaved into Myc-nick during the resolution of zymosan-induced peritonitis. . 47<br/> 2.4.4. Myc-nick localizes in the cytoplasm. 49<br/> 2.4.5. Myc-nick production is associated with efferocytosis 49<br/> 2.4.6. Myc-nick enhances efferocytic activity of macrophages. 54<br/> 2.4.7. Myc-nick promotes M2 polarization of macrophages during efferocytosis. . 57<br/> 2.4.8. Myc-nick potentiates efferocytosis through α-tubulin acetylation 60<br/> 2.4.9. Myc-nick induces LC3-associated phagocytosis (LAP). 64<br/> 2.5. Discussion 68<br/> <br/> Chapter III. 73<br/> 3.1. Abstract . 74<br/> 3.2. Introduction. 75<br/> 3.3. Materials and methods 78<br/> 3.3.1. Materials . 78<br/> 3.3.2. Methods. 78<br/> Animal experiment. 78<br/> Cell culture . 79<br/> Fractionation of cytosolic and nuclear extracts. 79<br/> Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis 80<br/> Electrophoresis mobility shift assay (EMSA) 81<br/> Colony formation assay 82<br/> Integrative network and correlation analysis 82<br/> Statistical analysis 83<br/> 3.4. Results 84<br/> 3.4.1. c-Myc expression is elevated in colitis-associated experimental carcinogenesis and human cancer cells . 84<br/> 3.4.2. RvD1 inhibits TNFα-induced overexpression of c-Myc in normal colon cells 87<br/> 3.4.3. RvD1 reduces the stability of c-Myc in colon cancer cells. 91<br/> 3.4.4. RvD1 inhibits c-Myc through interaction with the G-protein coupled formyl peptide receptor 2 (ALX/FPR2). 91<br/> 3.5. Discussion 97<br/> Conclusion and Perspectives . 100<br/> Reference 106<br/> Korean Abstract. 127<br/> Biographical Data 131<br/>Docto

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Publisher: 서울대학교 대학원
Year: 2018
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