Telomere-associated factor expression in replicative senescence of human embryonic lung fibroblasts

doi:10.3969/j.issn.2095-4344.2013.28.014

Abstract

Abstract:

BACKGROUND: Telomere-associated proteins will directly affect the function of telomeres, adjust the length of telomeric DNA, which are closely related with cell senescence and carcinogenesis.
OBJECTIVE: To find the key regulatory molecules in the cell senescence process through observing the telomere-associated factor expression in normal cell replicative senescence process.
METHODS: Based on established cell replicative senescence model, reverse transcription-PCR and western blot were used to detect the telomere-associated factor expression on the molecular and protein levels, including the telomere-associated factor human telomere binding protein 1, tankyrase 1, telomerase RNA, telomere protection protein 1 and P53 expressions in the human embryonic lung fibroblast replicative senescence.
RESULTS AND CONCLUSION: The results showed that with the cell senescence, transcription of human telomere binding protein 1 did not changed, while the protein expression of human telomere binding protein 1 was increased gradually and then decreased rapidly; mRNA and protein expressions of telomere protection protein 1 did not changed; with the human embryonic lung fibroblast replicative senescence, expression of telomere protection protein 1 was decreased gradually; with cell senescence, telomerase RNA component showed an increasing trend; protein expression of P53 did not changed. Human telomere binding protein 1, telomere protection protein 1 and telomerase RNA play an important role in cell senescence.

Key words: tissue construction, tissue construction and bioactive factors, replicative senescence, telomere binding protein 1, Tankyrase 1, telomere protection protein 1, telomerase RNA, P53, other grants-supported paper

CLC Number:

R318

Du Hua, Xu Xiao-yan, Hai Ling, Shi Ying-xu. Telomere-associated factor expression in replicative senescence of human embryonic lung fibroblasts[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(28): 5184-5190.

Figures/Tables 4

研究发现细胞的衰老伴随着端粒末端保护蛋白1的低表达，端粒末端保护蛋白1低表达可能是细胞复制性衰老的一个检测标志。人端粒结合蛋白1与端锚聚合酶1的在基因水平未发生变化，进一步检测了它们的蛋白表达情况。通过检测衰老过程中不同代龄人胚肺成纤维细胞发现人端粒结合蛋白1的蛋白表达水平随细胞的衰老呈增加的趋势，见图2A。表明人端粒结合蛋白1的蛋白表达量与衰老正相关。图2A中PD59的人端粒结合蛋白1低表达，推测是因为此时细胞开始进入细胞死亡阶段。前期细胞想通过高表达人端粒结合蛋白1来阻止细胞的衰老进程，但在细胞端粒不断缩短后，接近“Hayflick”极限，细胞启动凋亡机制，便不再高表达人端粒结合蛋白1的蛋白。通过检测衰老过程中不同代龄人胚肺成纤维细胞发现端锚聚合酶1的蛋白表达水平没有明显的变化，见图2B。表明端锚聚合酶1的蛋白水平在细胞复制性衰老过程中没有发生变化。端锚聚合酶1调节人端粒结合蛋白1与端粒DNA的结合，在细胞复制性衰老过程中未发生改变，说明端锚聚合酶1并未参与正常的衰老过程，可能只是在延长端粒或细胞复制过程中起作用。 2.3 端粒酶RNA在细胞衰老中的表达端粒酶RNA在1995年被发现，在各种细胞中普遍存在，且含量恒定。正常细胞无端粒酶活性但表达端粒酶RNA，端粒酶RNA会对细胞的复制性衰老起作用吗？实验通过检测衰老过程中不同代龄的人胚肺成纤维细胞端粒酶RNA的表达水平发现其mRNA表达水平随着细胞的衰老呈递增趋势，见图3。表明端粒酶RNA可能也参与细胞的复制衰老过程。说明衰老过程对于细胞造成巨大的生存压力，细胞为了缓解由于端粒丢失造成的应激，会激活促进端粒酶RNA的表达。"

References

[1]Xie Y, Yang D, He Q, et al. Zebrafish as a model system to study the physiological function of telomeric protein TPP1. PLoS One. 2011;6(2):e16440.

[2]Takai KK, Hooper S, Blackwood S, et al. In vivo stoichiometry of shelterin components. J Biol Chem. 2010;285(2):1457-1467.

[3]Takai KK, Kibe T, Donigian JR, et al. Telomere Protection by TPP1/POT1 Requires Tethering to TIN2. Molecular Cell. 2011;4 4: 647-659.

[4]Abreu E, Aritonovska E, Reichenbach P, et al. TIN2-tethered TPP1 recruits human telomerase to telomeres in vivo. Mol Cell Biol. 2010;30:2971-2982.

[5]Lundblad V. Taking the measuree. Nature. 2003;423:926-927.

[6]Blasco MA. Mammalian telomeres and telomerase:why they matter for cancer and aging. Eur J Cell Biol. 2003;82:441-446.

[7]Giannone RJ, McDonald HW, Hurst GB, et al. The Protein Network Surrounding the Human Telomere Repeat Binding Factors TRF1, TRF2, and POT1. PLoS ONE. 2010;5(8): e12407.

[8]Miller AS, Balakrishnan L, Buncher NA, et al. Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro. Cell Cycle. 2012;11(5):998-1007.

[9]Choi KH, Farrell AS, Lakamp AS, et al. Characterization of the DNA binding specificity of Shelterin complexes. Nucleic Acids Res. 2011;39(21):9206-9223.

[10]Martínez P, Blasco MA. Role of shelterin in cancer and aging. Aging Cell. 2010;9:653-666.

[11]Opresko PL, Kobbe Cv, Laine JP, et al. Telomere-binding Protein TRF2 Binds to and Stimulates the Werner and Bloom Syndrome Helicases. J Biol Chem. 2000;277(43):41110- 41119.

[12]Chiang YJ, Hsiao SJ, Yver D, et al. Tankyrase 1 and Tankyrase 2 Are Essential but Redundant for Mouse Embryonic Development. PLoS ONE. 2008;3(7): e2639.

[13]Ha GH, Kim HS, Go H, et al. Tankyrase-1 function at telomeres and during mitosis is regulated by Polo-like kinase-1-mediated phosphorylation. Cell Death Differ. 2012;19(2):321-332.

[14]Donigian JR, de Lange T. The role of the poly(ADP-ribose) polymerase tankyrase1 in telomere length control by the TRF1 component of the shelterin complex. J Biol Chem. 2007;282:22662-22667.

[15]Chang P, Coughlin M, Mitchison TJ. Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat Cell Biol. 2005;7:1133-1139.

[16]Gelmini S, Poggesi M, Distante V, et al. Tankyrase, a positive regulator of telomere elongation, is overexpressed in human breast cancer. Cancer Lett.2004;216:81-87.

[17]Flynn RL, Centore RC, O’Sullivan RJ, et al. TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA. Nature. 2011;471:532-536.

[18]Krüger AC, Raarup MK, Nielsen MM, et al. Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level. Eur Biophys J. 2010; 39(9):1343-1350.

[19]Choi YH, Lim JK, Jeong MW, et al. HnRNP A1 phosphorylated by VRK1 stimulates telomerase and its binding to telomeric DNA sequence. Nucleic Acids Res. 2012;40(17):8499-8518

[20]Ford LP, Wright WE, Shay JW. A model for heterogeneous nuclear ribonucleoproteins in telomere and telomerase regulation. Oncogene. 2002;21(4):580-583.

[21]Cairney CJ, Keith WN. Telomerase redefined: integrated regulation of hTR and hTERT for telomere maintenance and telomerase activity. Biochimie. 2008;90(1):13-23.

[22]Brault ME, Autexier C. Telomeric recombination induced by dysfunctional telomeres. Mol Biol Cell. 2011;22(2): 179-188.

[23]Ting NS, Pohorelic B, Yu Y, et al. The human telomerase RNA component, hTR, activates the DNA-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein A1. Nucleic Acids Res. 2009;37(18): 6105-6115.

[24]Fakhoury J, Marie-Egyptienne DT, Londoño-Vallejo JA, et al. Telomeric function of mammalian telomerases at short telomeres. J Cell Sci. 2010;123:1693-1704.

[25]Sexton AN, Youmans DT, Collins K. Specificity Requirements for Human Telomere Protein Interaction with Telomerase Holoenzyme. J Biol Chem. 2012;287(41):34455-34464.

[26]Cairney CJ, Keith WN. Telomerase redefined: integrated regulation of hTR and hTERT for telomere maintenance and telomerase activity. Biochimie. 2008;90(1):13-23.

[27]Itahana K, Dimri G, Campisi J. Regulation of cellular senescence by P53. Eur J Biochem. 2001;268:2784-2791.

[28]Feng Z, Lin M, Wu R. The Regulation of Aging and Longevity A New and Complex Role of p53. Genes Cancer. 2011; 2(4): 443-452.

[29]Harly CB, Futcher AB, Greider CW. Telomeres shorten during aging of human fibroblasts. Nature. 1990;345:4458-4604.

[30]Smogorzewska A , de-Lange T. Regulation of telomerase by telomeric proteins. Annu Rev Biochem. 2004;73: 177-208.

[31]Martínez P, Blasco MA. Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins. Nat Rev Cancer. 2011;11(3):161-176.

[32]van Steensel B, de Lange T. Control of telomere length by the human telomeric protein TRF1. Nature. 1997;385(6618): 740-743.

[33]Lee TH, Tun-Kyi A, Shi R, et al. Essential role of Pin1 in regulation of TRF1 stability and telomere maintenance. Nat Cell Biol. 2009;11(1): 97-105.

[34]Sfeir A, Kosiyatrakul ST, Hockemeyer D, et al. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell. 2009;138:90-103.

[35]Smith S, Giriat I, Schmitt A, et al. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science. 1998;282:1484-1487.

[36]Smith S, de Lange T. Tankyrase promotes telomere elongation in human cells. Curr Biol. 2000;10:1299-1302.

[37]Loayza D, De Lange T. POT1 as a terminal transducer of TRF1 telomere length control. Nature. 2003;423:1013-1018.

[38]Nandakumar J, Podell ER, Cech TR. How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA. Proc Natl Acad Sci U S A. 2010;107(2):651-656.

[39]Palm W, Hockemeyer D, Kibe T, et al. Functional dissection of human and mouse POT1 proteins. Mol Cell Biol. 2009;29: 471-482.

[40]Zhang DY, Wang HJ, Tan YZ. Wnt/β-catenin signaling induces the aging of mesenchymal stem cells through the DNA damage response and the p53/p21 pathway. PLoS One. 2011; 6(6):e21397.

[41]Demidenko ZN, Korotchkina LG, Gudkov AV, et al. Paradoxical suppression of cellular senescence by p53. Proc Natl Acad Sci U S A. 2010;107:9660-9664.

[42]Itahana K, Dimri G, Campisi J. Regulation of cellular senescence by P53. Eur J Biochem. 2001;268:2784-2791.