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Graduate School of Ajou University Department of Biomedical Sciences 4. Theses(Ph.D)

세포노화 및 TPA–유도 역노화에서 protein kinase C 동종효소의 역할규명

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dc.contributor.advisorIn Kyoung Lim-
dc.contributor.authorLee, Yun Yeong-
dc.date.accessioned2019-10-21T07:26:26Z-
dc.date.available2019-10-21T07:26:26Z-
dc.date.issued2016-02-
dc.identifier.other22087-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/18775-
dc.description학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2016. 2-
dc.description.tableofcontentsI. INTRODUCTION 1 II. MATERIALS AND METHODS 5 1. Cell culture 5 2. Senescence-associated-β-gal assay 5 3. Immunoprecipitation (IP) and immunoblot analyses 5 4. Cell fractionation 6 5. Immunocytochemistry 6 6. BrdU incorporation assay 6 7. Cell cycle analysis 7 8. Cell proliferation assay 7 9. Real-time PCR analysis 7 10. siRNA transfection 7 11. Statistical analysis 8 III. RESULTS 9 A. Nuclear translocation of pErk1/2 apart from cytoplasmic PEA-15 upon TPA treatment 9 B. Knockdown of PEA-15 expression induced pErk1/2 translocation to nuclei 12 C. Knockdown of PEA-15 and TPA treatment reduced senescence phenotype 15 D. Progression of G1/S phase by either TPA or siPEA-15 transfection 18 E. Increase of cell proliferation by either knockdown of PEA-15 or TPA treatment 20 F. Regulation of TPA-induced c-fos expression by nuclear Erk1/2 in HDF old cells 22 IV. DISCUSSION 24 V. CONCLUSION 26 Part I-B 27 I. INTRODUCTION 27 II. MATERIALS AND METHODS 34 1. Cell culture 34 2. Immunoblot (IB) analysis 34 3. Immunoprecipitation (IP) 35 4. Cells fractionation 35 5. Immunocytochemistry (ICC) 35 6. Two stage skin carcinogenesis 36 7. Immunofluorescence (IF) study 36 8. GST-pull down and in vitro kinase-immunoblot analyses 36 9. siRNA transfection 37 10. Plasmid transfection 37 11. Site-directed mutagenesis 37 12. RNA-seq analysis 37 12.1. Preprocessing and genome mapping 38 12.2. Quantifying gene expression and differential expressed gene analysis 38 12.3. Functional category analysis 39 13. Statistical analysis 39 III. RESULTS 40 A. Downregulation of PKCα reduces TPA response in HDF senescent cells to nuclear translocation of SA-pErk1/2 40 B. PKCβ1 regulates in vivo phosphorylation of PEA-15 at S104 residue which dissociates pErk1/2 from PEA-15 in HDF old cells 45 C. Nuclear translocation of Erk1/2 requires its activation and interaction with PKC upon TPA treatment 50 D. Swift degradation and delayed regeneration of PKCα expression allow senescent cells undergo reverse senescence upon TPA treatment 52 E. Loss of PKCα expression accompanies with acanthosis of epidermis in CD-1 mice by repetitive TPA treatment 56 F. RNA-seq analyses support reversal of senescence program after TPA treatment in HDF old cells 58 IV. DISCUSSION 69 V. CONCLUSION 73 Part II 74 I. INTRODUCTION 74 II. MATERIALS AND METHODS 78 1. Cell culture 78 2. Doxorubicin treatment 78 3. Senescence-associated-β-gal assay 78 4. Immunocytochemistry 79 5. BrdU incorporation assay 79 6. Real-time PCR analysis 79 7. siRNA transfection 80 8. Preparation of mitochondrial Fractions 80 9. Measurement of DNA Polymerase γ Activity 80 10. Electron microscopy analysis 81 11. Mitochondrial DNA copy-number analysis 81 12. ATP Determination 81 13. Intracellular ROS detection 81 14. Mitochondrial permeability potential assay 82 15. Mitochondrial respiration measurement 82 16. PKCζ kinase assay 82 17. Statistical analysis 82 III. RESULTS 83 A. Increased of dysfunctional mitochondria in replicative senescent human diploid fibroblast cells 83 B. Increase in punctuate marks of nucleoid along with BrdU incorporation and mitochondrial biogenesis in senescent cells 85 C. PKCζ is a key regulator of mitochondrial nucleoid and mitochondrial biogenesis in senescent HDF cells 88 D. Phenotypes in stress induced senescence was also observed by low-dose of Doxorubicin treatment to cells 90 E. Regulation of BrdU incorporation and mitochondrial biogenesis through PKCζ- LKB1-AMPK signaling pathway in stress induced senescent cells 92 IV. DISCUSSION 96 V. CONCLUSION & SCHEMETIC DIAGRAM 99 REFERENCES 100 국문요약 109-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title세포노화 및 TPA–유도 역노화에서 protein kinase C 동종효소의 역할규명-
dc.title.alternativeRole of protein kinase C isozymes in cellular senescence and reversal of senescence in response to 12-O-tetradecanoylphorbol-13 acetate (TPA) treatment in-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameYun Yeong Lee-
dc.contributor.department일반대학원 의생명과학과-
dc.date.awarded2016. 2-
dc.description.degreeDoctoral-
dc.identifier.localId739344-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000022087-
dc.subject.keywordSenescence Associated-pErk1/2-
dc.subject.keywordTPA (12-O-tetradecanoylphorbol-13-acetate)-
dc.subject.keywordReversal of senescence phenotype-
dc.subject.keywordPEA-15-
dc.subject.keywordHDF-
dc.subject.keywordPKCα-
dc.subject.keywordPKCβ1-
dc.subject.keywordubiquitination-
dc.subject.keywordDMBA-TPA-
dc.subject.keywordcarcinogenesis-
dc.subject.keywordacanthosis-
dc.subject.keywordskin-
dc.subject.keywordBrdU-
dc.subject.keywordPKCζ-
dc.subject.keywordTFAM-
dc.subject.keywordNRF1-
dc.subject.keywordLKB1-
dc.subject.keywordAMPK-
dc.subject.keywordNucleoid-
dc.subject.keywordMitochondiria.-
dc.description.alternativeAbstractCellular senescence plays an important role in biological processes such as development, aging and tumorigenesis, and it is a process of permanent growth arrest when cells lose ability to reactivate cell division cycle. One of the common molecular features of senescent cells is the failure of phospho-extracellular signal-regulated protein kinase 1/2 (pErk1/2) translocation to nuclei in response to growth factor stimulation. However, when senescent cells are treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), old cell morphology starts to change young cell like in addition to molecular markers of cellular senescence, such as increases of DNA synthesis, pRB hyperphosphorylation, reductions of p53, p21Sdi1/WAF1/CIP1 and SA-β-galactosidase activity. Mechanism of how 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using replicative senescence of HDF cells and DMBA-TPA induced carcinogenesis in CD-1 mice. Upon TPA treatment, PKCα and PKCβ1 played differentially in the nuclear translocation of senescence-associated pErk1/2, which regulates reversal of senescence; PKCα carried pErk1/2 to nuclei after freed from PEA-15pS104 by PKCβ1, and then rapidly degraded by ubiquitination. MAPK docking motif and kinase activity in PKCα were required to carry pErk1/2. Moreover, repetitive application of TPA on mouse skin revealed significant loss of PKCα expression along with acanthosis in epidermis and hair follicle, indicating that downregulation of PKCα was accompanied with epidermal proliferation. The above observations were further supported by the RNA-seq analyses in HDF old cells: TPA-mediated PKCα degradation allows pErk1/2 to be free to promote cell proliferation in both senescence and carcinogenesis, emphasizing the role of PKC isozymes and cytoplasmic pErk1/2 in the regulation of cellular senescence. Mitochondrial dysfunction is linked between age-related accumulation of oxidative damage and alterations of physiological function associated with senescence. Recent studies suggest that mitochondrial metabolism is upregulated in oncogene induced senescent cells to meet with metabolic demand of cytokine production. In addition, a partial uncoupling of oxidative phosphorylation in mitochondria has been reported in the senescence fibroblast cells; thus ATP production is insufficient despite increased oxygen consumption. We report herein increased mass and DNA contents of mitochondria in the replicative HDF cells along with mitochondrial hyperfusion, and elevated expression of OXPHOS complex 4 and 5 proteins than those of the young cells. Furthermore, increased 5-bromo-2'-deoxyuridine (BrdU) incorporation at the mitochondrial nucleoid along with mitochondrial transcription factors A (TFAM). Nevertheless, we observed that mitochondria dysfunction was increased via alteration of ROS level, integrity of membrane potential, mitochondria cristae structure and ATP content. To explore signaling pathways regulating the phenotypes, we analyzed the mitochondrial TFAM protein expression which was found to be significantly increased in old cells. Up regulation of TFAM was accompanied with increased PGC-1α and NRF1 expressions through the activations of LKB1 and AMPK due to increased activity of PKCζ in old cells. These signaling pathways are important in regulating the mitochondrial encoding OXPHOS complex subunit genes and respiration as well as ATP generation, evidenced by employing siRNA against PKCζ. All of the findings were further confirmed in the doxorubicin-induced premature senescence of young HDF cells. These datas indicate that continuous increase of BrdU positive cells and mitochondrial biogenesis is regulated by LKB1, AMPK and PKCζ, despite higher ROS accumulation in stress induced senescence model. In summary, the activation of mitochondrial biogenesis pathway via PKCζ-LKB1-AMPK in senescent cells might be due to the compensation of mitochondrial dysfunction, which stimulated maintaining of nucleoid structure and TFAM activity via PGC-1α and NRF1 increased by LKB1-AMPK-PKCζ signal pathway. Our present study suggests a new concept about mitochondrial function in old cells and offers a plausible explanation on the role of mitochondrial hyperfunction in senescence, being a survival reaction when exposed to lower energy condition and cell stress.-
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