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세포노화 및 TPA–유도 역노화에서 protein kinase C 동종효소의 역할규명
- Alternative Title
- Role of protein kinase C isozymes in cellular senescence and reversal of senescence in response to 12-O-tetradecanoylphorbol-13 acetate (TPA) treatment in
- Author(s)
- Lee, Yun Yeong
- Alternative Author(s)
- Yun Yeong Lee
- Advisor
- In Kyoung Lim
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2016-02
- Language
- eng
- Keyword
- Senescence Associated-pErk1/2; TPA (12-O-tetradecanoylphorbol-13-acetate); Reversal of senescence phenotype; PEA-15; HDF; PKCα; PKCβ1; ubiquitination; DMBA-TPA; carcinogenesis; acanthosis; skin; BrdU; PKCζ; TFAM; NRF1; LKB1; AMPK; Nucleoid; Mitochondiria.
- Alternative Abstract
- Cellular 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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