| Background and ObjectivesThe pathogenesis of skin cancer has been a spot in clinical research. Previous studies have shown that ultraviolt B is one of the reasons for UV-induced skin injury, even in skin cancers.95% UVB reached in skin surface absorbed by keratinocytes could destroy the cell’s biological function, which would induced cell death and apoptosis through different ways, which include direct DNA damage, production of free radial and peroxidation acting on the cell membrane and mitochondrial membrane, activation of death receptors on the surface of the cell membrane and degradation of neural sphingomyelinase to sphingomyelin, which resulted in synthesis of increased second messenger ceramide and its derivatives.PI3K/Akt/mTOR is a major signal transduction pathway to mediate cell proliferation, apoptosis and differentiation. A large number of studies have shown that UVB-inhibited cell apoptosis would promote tumor cell proliferation and invasive through activation of PI3K/Akt/mTOR pathways. Akt plays an important role in activation of PI3K/Akt/mTOR cell signal transduction pathways. Akt (Ser-473) and Akt (Thr-308) phosphorylation are the necessary step for Akt activation. However, whether UVB induced Akt activation is related to two sites phosphorylation is not fully clear at present.UVB would cause skin cancer through direct or indirect damaged DNA and induced gene mutation. Repair of DNA damage play an important role in maintaining the genetic stability. Non-homologous end-joining (NHEJ) is main DNA damage repair in mammals. DNA-dependent protein kinase (DNA-PK) is a key composition of NHEJ. DNA-PK is consistes of DNA-dependent protein Kinase catalytic subunit (DNA-PKcs) and two regulatory subunits, Ku70 and Ku80. Many Studies have shown that some special stimulation, such as ionizing radiation, could induce activation of DNA-PKcs and activated Akt. Meanwhile, mammalian Target of Rapamycin complex 2(mTORC2), is one complex of mammalian Target of Rapamycin(mTOR) could also activated Akt. DNA-PKcs and mTORC2 are regard as Akt kinase. However, whether UVB could induce DNA-PKcs and mTORC2 activation, DNA-PKcs and mTORC2 further activated Akt is still not fully understood. Therefore, in the study the mechanisms of UVB activation of PI3K/Akt/mTOR pathway and DNA-PKcs and mTORC2 activated by UVB activated Akt have been further investigated, which provide an important target for prevention and treatment of skin cancer.Part 1. Investigation on the mechanisms of UVB activation of PI3K/Akt/mTOR pathwayMethodsPrimary human epidermal keratinocytes were irradiated by UVB of different doses (0, 5,15,30mJ/cm2) for 1h, the expressions of phosphorylated Akt Ser-473, Akt Thr-308, mTORC1, GSK3βs9, S6K (Thr389),4EBP-1 and mTORC2 were tested by Western blots. The primary human epidermal keratinocytes were irradiated by UVB(30mJ/cm2) for 0min,15min,30min, 1h, or 2h. The expression of these factors was tested by Western blots again.Results1. When the keratinocytes was irradiated by different doses of UVB, the results of Western blots have shown that the expressions of p-Akt (Thr308), p-Akt (Ser473), p-GSK3β, p-mTOR (S2448), p-S6K (Thr389), p-S6 (Ser235/236) and p-Rictor (Tyr1135) were increased in dose-dependent manner, and were highest after UVB(30mJ/cm2) irradiation (P<0.05)2. When the keratinocytes was irradiated by UVB (30 mJ/cm2), the results of Western blots have shown that the expressions of p-Akt (Thr308), p-Akt (Ser473), p-GSK3β, p-mTOR (S2448),p-S6K (Thr389), p-S6 (Ser235/236) and p-Rictor (Tyr1135) were increased in time-dependent manner. The expressions of p-Akt (Thr308), p-Akt (Ser473), p-S6K (Thr389) were highest after UVB irradiation for 2 hours (P<0.05), and the expressions of p-GSK3β, p-mTOR (S2448), p-S6 (Ser235/236), p-4E-BP1, p-Rictor (Tyr1135) were highest after UVB irradiation for 1 hour (P<0.05)ConclusionsUVB activates PI3K/Akt/mTOR pathway through Phosphorylated Akt (Ser473) and Akt(Thr308).Part 2. Investigation on the mechanisms of DNA-PKcs and SIN1 mediating UVB inducing PI3K/Akt/Mtor activationMethods1. The keratinocytes were irradiated by different doses of UVB (0,5,15,30mJ/cm2) The expressions of phosphorylated DNA-PKcs Thr-2647 and Thr-2606 were tested by Western blots.2. Primary skin keratinocytes and HaCat cells were transfected with scramble and two distinct DNA-PKcs siRNA(-1 and -2), primary skin keratinocytes were transfected with pSV2 neo plasmid including T2609A DNA-PKcs. These cells were irradiated by UVB (30mJ/cm2). The expressions of phosphorylated Akt Ser-473, Akt Thr-308 and Erkl/2 were tested by Western blots.3. The pretreated keratinocytes with NU7026(DNA-PKcs inhibitor) were irradiated by UVB (30mJ/cm2).The expressions of phosphorylated Akt Ser-473, S6 (Ser-235/236), Erkl/2 were tested by Western blots.4. The pretreated keratinocytes with NU7026 were irradiated by UVB (30mJ/cm2). MTT assay was used to test cell survival. Cell apoptosis was analyzed by Histone-DNA ELISA assay and Annexin V FACS assay.5. WT and DNA-PKcs KO MEFs were irradiated by UVB(30mJ/cm2). The expressions of phosphorylated Akt Ser-473, Akt Thr-308 were tested by Western blots.6. Primary skin keratinocytes transfected with scramble and SIN1 siRNA were irradiated by UVB (30mJ/cm2). The expressions of phosphorylated Akt Ser-473, Akt Thr-308, Erkl/2 were tested by Western blots.Resultsl.When the keratinocytes were irradiated by different dose of UVB, the results of western blots have shown that the expression of p-DNA-PKcs(Thr2647) and p-DNA-PKcs(Thr2609) was increased in dose-dependent manner,and were highest after UVB(45 mJ/cm2) irradiation (P<0.05)2.After UVB (30mJ/cm2) irradiated primary skin keratinocytes and HaCat cells that were transfected with DNA-PKcs siRNA(-1 and -2) and the pSV2 neo plasmid including T2609A DNA-PKcs, the result of western blots have shown that the expressions of p-Akt(Ser473) decreased significantly(p<0.05), while the expressions of p-Akt (Ser308) did not significantly change(p>0.05).3. When the pretreated keratinocytes with NU7026 irradiated by UVB (30mJ/cm2), the result of Western blots show that the expression of p-Akt Ser-473 decreased significantly(p<0.05), while the expressions of p-S6 (Ser235/236) and p-Erkl/2 did not significantly change(p>0.05).4. When the pretreated keratinocytes with NU7026 irradiated by UVB (5,15,30 or 40mJ/cm2), the survival rates of these cells were decreased significantly and the apoptosis rates of these cells were increased significantly (p<0.05).5, WT, DNA-PKcs KO and SIN1 KO (-/-)MEFs were irradiated with UVB (UV,30 mJ/cm2) and cultured for 1 hr, the result of Western-blots show that the expressions of p-Akt Ser-473 decreased significantly(p<0.05), while the expressions of p-Akt Ser-308 did not significantly change(p>0.05).6. After UVB (30mJ/cm2) irradiated primary skin keratinocytes transfected with DNA-PKcs siRNA, the result of Western-blots show that the expressions of p-Akt Ser-473 decreased significantly(p<0.05), while the expressions of p-Akt Ser-308 did not significantly change(p>0.05).ConclusionsUVB activates DNA-PKcs through Phosphorylated DNA-PKcs(Thr2647) and DNA-PKcs(Thr2609).when activation of DNA-PKcs was inhibited,or DNA-PKcs(Thr2609) mutations,Akt (Ser473) phosphorylation could be inhibited. Which promoted cell apoptosis and inhibited cell proliferation.Part 3. Investigation on the mechanisms of UVB inducing DNA-PKcs-SINl complex formationMethods1. Primary skin keratinocytes were divided into control group and experimental groups, the cells in experimental groups were treated with NU7026 and were transfected with the pSV2 neo plasmid including T2609A DNA-PKcs and DNA-PKcs siRNA(-2). Then these cells were irradiated with UVB (30mJ/cm2). The expression of DNA-PKcs, SIN1, Ku80, mTOR were tesed by Co immuneprecipitation(Co-IP),2. Primary skin keratinocytes were irradiated with UVB(30mJ/cm2) and harvested at various time (Omin,15min,30min or 1h). The expression of DNA-PKcs, SIN1 and Akt in nuclear and cytosol were tested by Western blots.Results1.After UVB (30mJ/cm2) irradiated primary skin keratinocytes that were protreated with NU7026, the results of Co-IP show that in control group, the expression of SIN1 were positive when DNA-PKcs coprecipitated with SNI1,while the expression of DNA-PKcs were positive when SMI coprecipitated with DNA-PKcs.But in experimental groups, there were no expression of SIN1 when DNA-PKcs coprecipitated with SNI1, while there were no expression of DNA-PKcs when SNI1 coprecipitated with DNA-PKcs.2.The results of Western blots show that the expressions of DNA-PKcs, SIN1 and Akt were increased in cytosol at 10min,15min and 30min after UVB (30mJ/cm2) irradiated primary skin keratinocytes significantly(p<0.05).ConclusionsUVB induced DNA-PKcs/SIN1 complex formation in cytosol. DNA-PKcs/SIN1 complex mediated UVB induced Akt activation.Part 4. Investigation on the mechanisms of EGFR mediating UVB inducing DNA-PKcs/SIN1 complex formation and Akt activationMethods1. Primary skin keratinocytes were treated with AG1478(EGFR inhibitor), and were irradiated by UVB(30mJ/cm2). The expressions of phosphorylated DNA-PKcs, mTOR and Akt-Ser473 were tested by Western blots. The expressions of DNA-PKcs and SIN1 were tested by Co-IP.2. EGFR KO MEFs were irradiated with UVB(30mJ/cm2). The expressions of phosphorylated DNA-PKcs, mTOR nad Akt-Ser473 were tested by Western blots. The expressions of DNA-PKcs and SIN1 were tested by Co-IP.ResultsAfter UVB (30mJ/cm2) irradiated primary skin keratinocytes that were pretreated with NU7026 and EGFR-KO-MEFs,the results of Western blots show that the expression of p-Akt (Ser-473) and p-mTOR were decreased significantly(p<0.05).The results of Co-IP show that there were no expression of SIN1 when DNA-PKcs coprecipitated with SNI1, while there were no expression of DNA-PKcs when SNI1 coprecipitated with DNA-PKcs(p>0.05).ConclusionsEGFR mediates UVB induced DNA-PKcs/SINl association.All of the results have show that UVB could induce the function of DNA-PKcs-SINl complex, further would activate Akt through phosphorylation of Akt (Ser-473). Which could promoted cell apoptosis and inhibited cell proliferation.Statistical AnalysisThe results were presented as mean±SD. Data were analyzed by one-way ANOVA followed by using SPSS 15.0 software. Significance was chosen as p<0.05. |
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