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배양된 사람 각막내피세포에서 Cyclosporine A로 유도된 노화의 효과

Effect of Cyclosporine A-induced Senescence on Cultured Human Corneal Endothelial Cells

신영주|정태영|장윤경
대한안과학회 (The Korean Ophthalmological Society)|2020.09|대한안과학회지 (Journal of The Korean Ophthalmological Society)|제61권 제9호|pp. 999-1009

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SenescenceMitochondrial membrane potentialCyclosporine AHuman corneal endothelial cellsMitochondrial oxidative stress
주제분류안과학

초록

목적: 생체 외에서 배양된 사람 각막내피세포에서 cyclosporine A (CsA)로 인한 노화의 영향에 대해 알아보고자 하였다. 대상과 방법: 사람 각막내피세포를 배양하고 0-100 μM CsA와 incubation을 시행하였다. 노화 연관 베타 갈락토시다제(senescenceassociated β-Galactosidase, SA-β-gal) 염색을 시행하였다. WST-8 분석 키트를 사용하여 미토콘드리아 탈수소효소의 활성을 평가하고, JC-1 dye를 이용하여 미토콘드리아막 전위(ΔΨm)를 측정하였다. 세포 내와 미토콘드라아에서 활성 산소 형성을 측정하였다. 세포 내와 미토콘드리아의 칼슘 수치를 각각 Fluo-4와 Rhod-2를 통해 측정하였다. 단백질 발현은 웨스턴 블롯(western blot)에 의해 평가되었다. 결과: 농도 의존적으로 CsA는 SA-β-gal 양성 세포의 비율을 증가시켰고(p=0.003) 미토콘드리아 탈수소효소의 활성과 ΔΨm을 감소시켰다(p=0.029, p=0.004). 세포 내 및 미토콘드리아의 활성 산소는 CsA와 배양하는 동안 증가하였다(p=0.005). 100 μM의 CsA에서 미토콘드리아 칼슘 수치가 높아진 반면(p=0.001), 세포 내 칼슘 수치는 낮아졌다(p=0.029). CsA는 GSK3β와 ERK1/2를 활성화시키고 ZO-1 발현을 감소시켰다. 결론: 사람 각막내피세포에서 CsA는 산화 스트레스의 유도와 미토콘드리아, GSK3β 및 ERK1/2 경로를 통해 노화를 유도한다. 따라서 고농도의 CsA 사용시 주의하여 사용하여야 한다.

Abstract

Purpose: To investigate the in vitro effect of cyclosporine A (CsA)-induced senescence on human corneal endothelial cells (HCECs). Methods: HCECs were cultured and incubated with 0-100 μM CsA. Senescence-associated β–galactosidase (SA-β-gal) staining was performed. Mitochondrial dehydrogenase activity was assessed using a WST-8 assay kit and mitochondrial membrane potential (ΔΨm) was measured using JC-1 dye. Intracellular and mitochondrial formation of reactive oxygen species (ROS) was measured with 2’,7’-dichlorodihydrofluorescein diacetate and MitoSOX probes. Intracellular and mitochondrial calcium levels were measured using Fluo-4 and Rhod-2, respectively. Protein expression was evaluated by Western blotting. Results: CsA increased the percentage of SA-β-gal-positive cells (p = 0.003) and decreased mitochondrial dehydrogenase activity and ΔΨm in a dose-dependent manner (p = 0.029, p = 0.004). Intracellular and mitochondrial ROS levels increased during incubation with CsA (p = 0.005). CsA at 100 μM increased mitochondrial calcium levels (p = 0.001), whereas intracellular calcium levels decreased at 100 μM CsA (p = 0.029). CsA activated GSK3β and ERK1/2 and reduced ZO-1 expression. Conclusions: CsA induces senescence in HCECs through oxidative stress and via mitochondria-, GSK3β-, and ERK1/2-dependent pathways. Thus, concentrations of CsA should be monitored.

참고문헌 (53)

  1. Joyce NC, "Proliferative capacity of the corneal endothelium", Prog Retin Eye Res, 22, pp.359~389 (2003)
  2. Shin YJ, "Rapamycin reduces reactive oxygen species in cultured human corneal endothelial cells", Curr Eye Res, 36, pp.1116~1122 (2011)
  3. Hatou S, "Role of insulin in regulation of Na+-", K+-dependent ATPase activity and pump function in corneal endothelial cells, 2010, pp.51, 3935~3942
  4. Waring GO 3rd, "The corneal endothelium. Normal and pathologic structure and function", Ophthalmology, 89, pp.531~590 (1982)
  5. Whikehart DR, "The inhibition of sodium, potassium-stimulated ATPase and corneal swelling : the role played by polyols", J Am Optom Assoc, 66, pp.331~333 (1995)
  6. Chifflet S, "Early and late calcium waves during wound healing in corneal endothelial cells", Wound Repair Regen, 20, pp.28~37 (2012)
  7. Engler C, "Unfolded protein response in fuchs endothelial corneal dystrophy : a unifying pathogenic pathway", Am J Ophthalmol, 149, pp.194~202.e2 (2010)
  8. Bourne WM, "Central corneal endothelial cell changes over a ten-year period", Invest Ophthalmol Vis Sci, 38, pp.779~782 (1997)
  9. Afshari NA, "Clinical study of Fuchs corneal endothelial dystrophy leading to penetrating keratoplasty : a 30-year experience", Arch Ophthalmol, 124, pp.777~780 (2006)
  10. Toussaint O, "Aging as a multi-step process characterized by a lowering of entropy production leading the cell to a sequence of defined stages. II. Testing some predictions on aging human fibroblasts in culture", Mech Ageing Dev, 65, pp.65~83 (1992)
  11. Toussaint O, "Stress-induced premature senescence. Essence of life, evolution, stress, and aging", Ann N Y Acad Sci, 908, pp.85~98 (2000)
  12. Mammone T, "Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures", Cell Biol Int, 30, pp.903~909 (2006)
  13. Chen JH, "Methods of cellular senescence induction using oxidative stress", Methods Mol Biol, 371, pp.179~189 (2007)
  14. Prigione A, "The senescence-related mitochondrial", oxidative stress pathway is repressed in human induced pluripotent stem cells, 2010, pp.28, 721~733
  15. O'Connell S, "Cyclosporine A-induced oxidative stress in human renal mesangial cells : a role for ERK 1", 2MAPK signaling, 2012, pp.126, 101~113
  16. Werneck MB, "Cyclosporin A inhibits colon cancer cell growth independently of the calcineurin pathway", Cell Cycle, 11, pp.3997~4008 (2012)
  17. Flanagan WM, "Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A", Nature, 352, pp.803~807 (1991)
  18. Tatlipinar S, "Topical ciclosporin in the treatment of ocular surface disorders", Br J Ophthalmol, 89, pp.1363~1367 (2005)
  19. Belin MW, "Topical cyclosporine in high-risk corneal transplants", Ophthalmology, 96, pp.1144~1150 (1989)
  20. Pérez-Rico C, "05% cyclosporine A on corneal endothelium in patients with dry eye disease", Int J Ophthalmol, 6, pp.471~474 (2013)
  21. Shin YJ, "Cysteamine suppresses human peripheral blood mononuclear cells--human corneal endothelial cell reaction via reactive oxygen species reduction", Mol Vis, 17, pp.3371~3378 (2011)
  22. Kim E, "The effects of different culture media on human corneal endothelial cells", Invest Ophthalmol Vis Sci, 55, pp.5099~5108 (2014)
  23. Severino J, "Is beta-galactosidase staining a marker of senescence in vitro and in vivo?", Exp Cell Res, 257, pp.162~171 (2000)
  24. He Y, "Pro370Leu mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells", Mol Vis, 15, pp.815~825 (2009)
  25. Ardón F, "Mitochondrial inhibitors activate influx of external Ca(2+)in sea urchin sperm", Biochim Biophys Acta, 1787, pp.15~24 (2009)
  26. Choi WS, "Cytotoxicity of ganciclovir on cultured human corneal endothelial cells", Antivir Ther, 18, pp.813~820 (2013)
  27. Kwak IH, "Nuclear accumulation of globular actin as a cellular senescence marker", Cancer Res, 64, pp.572~580 (2004)
  28. Mao Z, "Replicatively senescent cells are arrested in G1 and G2 phases", Aging(Albany NY), 4, pp.431~435 (2012)
  29. Di Lernia V, "Effectiveness and safety of cyclosporine in pediatric plaque psoriasis : a multicentric retrospective analysis", J Dermatolog Treat, 27, pp.395~398 (2016)
  30. Caires A, "Endothelin-1 receptor antagonists protect the kidney against the nephrotoxicity induced by cyclosporine-A in normotensive and hypertensive rats", Braz J Med Biol Res, 51, pp.e6373~ (2017)
  31. Wu Q, "Mechanism of cyclosporine A nephrotoxicity : oxidative stress, autophagy, and signalings", Food Chem Toxicol, 118, pp.889~907 (2018)
  32. Koppelstaetter C, "Effect of cyclosporine, tacrolimus and sirolimus on cellular senescence in renal epithelial cells", Toxicol In Vitro, 48, pp.86~92 (2018)
  33. Justo P, "Intracellular mechanisms of cyclosporin A-induced tubular cell apoptosis", J Am Soc Nephrol, 14, pp.3072~3080 (2003)
  34. Mandavilli BS, "Mitochondrial DNA repair and aging", Mutat Res, 509, pp.127~151 (2002)
  35. Lin MT, "Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases", Nature, 443, pp.787~795 (2006)
  36. Galluzzi L, "Mitochondrial control of cellular life, stress, and death", Circ Res, 111, pp.1198~1207 (2012)
  37. Joshi DC, "Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons", J Vis Exp, 51, pp.2704~ (2011)
  38. Zoratti M, "The mitochondrial permeability transition", Biochim Biophys Acta, 1241, pp.139~176 (1995)
  39. Peng TI, "Oxidative stress caused by mitochondrial calcium overload", Ann N Y Acad Sci, 1201, pp.183~188 (2010)
  40. van der Toorn M, "Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential", FEBS J, 274, pp.3003~3012 (2007)
  41. Simpson PB, "High density distribution of endoplasmic reticulum proteins and mitochondria at specialized Ca2+ release sites in oligodendrocyte processes", J Biol Chem, 272, pp.22654~22661 (1997)
  42. Babcock DF, "Mitochondrial participation in the intracellular Ca2+ network", J Cell Biol, 136, pp.833~844 (1997)
  43. Robb-Gaspers LD, "Integrating cytosolic calcium signals into mitochondrial metabolic responses", EMBO J, 17, pp.4987~5000 (1998)
  44. Petit-Paitel A, "Involvment of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP", MPP-treated neurons, 2009, pp.4, e5491~
  45. Patel S, "Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword?", Biochem Soc Trans, 32, pp.803~808 (2004)
  46. Berzal S, "GSK3, snail, and adhesion molecule regulation by cyclosporine A in renal tubular cells", Toxicol Sci, 127, pp.425~437 (2012)
  47. Kim HS, "Cyclosporine A induces apoptotic and autophagic cell death in rat pituitary GH3 cells", PLoS One, 9, pp.e108981~ (2014)
  48. Deng Q, "High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts", J Biol Chem, 279, pp.1050~1059 (2004)
  49. Cagnol S, "ERK and cell death : mechanisms of ERK-induced cell death--apoptosis, autophagy and senescence", FEBS J, 277, pp.2~21 (2010)
  50. Rusnak F, "Calcineurin : form and function", Physiol Rev, 80, pp.1483~1521 (2000)
  51. Krouwer VJ, "Endothelial cell senescence is associated with disrupted cell-cell junctions and increased monolayer permeability", Vasc cell, 4, pp.12~ (2012)
  52. Yanai R, "Upregulation of ZO-1 in cultured human corneal epithelial cells by a peptide(PHSRN)corresponding to the second cell-binding site of fibronectin", Invest Ophthalmol Vis Sci, 50, pp.2757~2764 (2009)
  53. Lallemand F, "Cyclosporine A delivery to the eye : a comprehensive review of academic and industrial efforts", Eur J Pharm Biopharm, 117, pp.14~28 (2017)

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