한국발생생물학회지:발생과생식 (Development and Reproduction)

  • 제15권4호
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  • Pages.373-383
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  • 2011
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  • 2465-9525(pISSN)
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  • 2465-9541(eISSN)
한국발생생물학회 (The Korean Society of Developmental Biology)
권호 표지

Tributyltin에 의한 흰쥐 흉선 내 상피세포의 지방세포 유도와 세포자연사 증가

Tributyltin Induces Adipogenesis and Apoptosis of Rat Thymic Epithelial Cells

  • 이효진 (서울여자대학교 자연과학대학 생명환경공학과) ;
  • 이아라 (서울여자대학교 자연과학대학 생명환경공학과) ;
  • 안보람 (서울여자대학교 자연과학대학 생명환경공학과) ;
  • 전은제 (서울여자대학교 자연과학대학 생명환경공학과) ;
  • 정예지 (서울여자대학교 자연과학대학 생명환경공학과) ;
  • 양현원 (서울여자대학교 자연과학대학 생명환경공학과)
  • Lee, Hyo-Jin (Dept. of Bioenvironmental Technology, College of Natural Sciences, Seoul Women's University) ;
  • Lee, A-Ra (Dept. of Bioenvironmental Technology, College of Natural Sciences, Seoul Women's University) ;
  • Ahn, Bo-Ram (Dept. of Bioenvironmental Technology, College of Natural Sciences, Seoul Women's University) ;
  • Jeon, Eun-Je (Dept. of Bioenvironmental Technology, College of Natural Sciences, Seoul Women's University) ;
  • Jeong, Ye-Ji (Dept. of Bioenvironmental Technology, College of Natural Sciences, Seoul Women's University) ;
  • Yang, Hyun-Won (Dept. of Bioenvironmental Technology, College of Natural Sciences, Seoul Women's University)
  • 투고 : 2011.12.01
  • 심사 : 2011.12.18
  • 발행 : 2011.12.31
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초록

성 호르몬과 유사한 기능을 하는 것으로 알려진 내분비교란물질은 흰쥐 흉선세포에 세포자연사를 일으켜 흉선의 기능을 감소시키는 것으로 보고되고 있으나, 그 작용 기전에 대해서는 잘 알려져 있지 않다. 따라서 본 연구에서는 내분비 교란물질 중 하나인 Tributyltin(TBT)를 흰쥐에 투여한 후 흉선의 상피세포가 지방세포로 분화되는지를 조사하고, 이로 인한 T 세포의 세포자연사와 연관성을 조사함으로써 TBT가 흉선기능에 미치는 영향을 알아보고자 하였다. 3주령 된 암컷 흰쥐에 각각 TBT 1, 10, 25 mg/kg/day를 1주일 동안 경구 투여한 후, 흉선을 획득하여 무게와 세포 수를 측정하였고, 파라핀 절편으로 H&E 염색, TUNEL 분석을 수행하였다. 또한 real-time PCR 방법으로 상피세포 표지 유전자들, 지방세포유도 유전자들과 세포자연사 관련 유전자들을 비교 분석하였으며, 유세포 분석기를 통해 세포자연사 정도를 조사하였다. 흉선의 무게와 세포 수는 대조군과 비교하여 TBT의 농도가 높을수록 유의하게 감소하였다. H&E 염색 결과, TBT의 농도가 증가할수록 흉선의 크기가 감소하고 피질과 수질의 경계가 흐려짐을 관찰하였다. TUNEL 염색 결과에서는 대조군에 비해 TBT를 투여한 군에서 세포자연사가 증가하였으며, 유세포 분석결과에서도 TBT 농도 의존적으로 세포자연사가 증가하였다. Real-time PCR 결과, 상피세포 표지 유전자인 EVA, IL-7, AIRE, KGF는 TBT의 농도가 증가할수록 발현양이 유의하게 감소한 반면, 지방세포 유도와 관련된 유전자인 $PPAR{\gamma}$, aP2, PEPCK, CD36은 TBT의 농도가 증가할수록 발현양이 유의하게 증가하였다. 세포자연사와 관련된 유전자인 $TNF{\alpha}$, TNFR1도 TBT의 농도가 증가할수록 발현양이 유의하게 증가하였다. 위의 결과를 종합하여 볼 때, 내분비교란물질인 TBT는 흉선 상피세포에 직접적으로 작용하여 지방세포로 분화 유도시킴으로써 흉선의 기능을 상실케 만들면서 T 세포의 발달에 영향을 미치는 것으로 보인다. 이러한 결과는 지속적으로 노출되는 TBT가 흉선의 기능을 떨어뜨림으로써 면역력 저하를 유발시킬 수 있음을 제시하고 있다.

Tributyltin (TBT) is one of endocrine disrupters which are known as having similar function to sex steroid hormone inducing apoptosis in various tissues of rodents. Recently, it has been reported that TBT induces apoptosis in thymus causing the decreased thymic function, but little is known about the mechanism. To elucidate the mechanism, three-week-old SD female rats were orally administrated with TBT 1, 10, and 25 mg per body weight (kg) and sesame oil as a control for 7 days. On day 8, the thymi were obtained and weighed, and then the number of thymocytes was counted. We also performed H&E staining, TUNEL assay, and Annexin V flow cytometric analysis to examine the apoptosis rates and the structure in the thymus. Next, we investigated the adipogenesis and apoptosis-related mRNA expression levels in the thymi by real-time PCR. The thymic weight and the number of thymocytes were decreased by TBT in a dose-dependent manner. As a result of the H&E staining, the boundary between cortical and medullary area was blurred in the thymi of TBT treated rats compared to those of controls. In the results of TUNEL assay and Annexin V flow cytometric analysis, apoptosis rates in the thymus were increased after TBT treatment. The expression levels of thymic epithelial cell marker genes such as EVA, KGF, AIRE, and IL-7 were significantly decreased in the thymi of TBT treated rats, but $PPAR{\gamma}$, aP2, PEPCK, and CD36 were significantly increased. The expression of $TNF{\alpha}$ and TNFR1 as apoptosis-related genes also was significantly increased after TBT treatment. The present study demonstrates that TBT can increase the expression of adipogenesis and apoptosis-related genes leading to apoptosis in the thymus. These results suggest that the increased adipogenesis of thymus by TBT exposure might induce apoptosis in the thymus resulting in a loss in thymic immune function.

키워드

참고문헌

  1. Berger J, Moller DE (2002) The mechanisms of action of PPARs. Annu Rev Med 53:409-435. https://doi.org/10.1146/annurev.med.53.082901.104018
  2. Blaber SJM (1970) The occurrence of a penis-like outgrowth behind the right tentacle in spent females of Nucella lapillus. J Mollus Stud 39:231-233. https://doi.org/10.1093/oxfordjournals.mollus.a065097
  3. Carfi M, Croera C, Ferrario D, Campi V, Bowe G, Pieters R, Gribaldo L (2008) TBTC induces adipocyte differentiation in human bone marrow long term culture. Toxicology 249:11-18. https://doi.org/10.1016/j.tox.2008.03.025
  4. Chen Y, Zuo Z, Chen S, Yan F, Chen Y, Yang Z, Wang C (2008) Reduction of spermatogenesis in mice after tributyltin administration. Toxicology 251:21-27. https://doi.org/10.1016/j.tox.2008.06.015
  5. Danilenko DM (1999) Preclinical and early clinical development of keratinocyte growth factor, an epithelialspecific tissue growth factor. Toxicol Pathol 27:64-71. https://doi.org/10.1177/019262339902700113
  6. Erickson M, Morkowski S, Lehar S, Gillard G, Beers C, Dooley J, Rubin JS, Rudensky A, Farr AG (2002) Regulation of thymic epithelium by keratinocyte growth factor. Blood 100:3269-3278. https://doi.org/10.1182/blood-2002-04-1036
  7. Fawthrop DJ, Boobis AR, Davies DS (1991) Mechanisms of cell death. Arch Toxicol 65:437-444. https://doi.org/10.1007/BF01977355
  8. Febbraio M, Hajjar DP, Silverstein RL (2001) CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest 108:785-791. https://doi.org/10.1172/JCI14006
  9. Feng J, Han JH, Pearce SF, Silverstein R, Gotto AM, Hajjar DP, Nicholson AC (2000) Induction of CD36 expression by oxidized LDL and IL-4 by a common signaling pathway dependent on protein kinase C and PPAR-gamma. J Lipid Res 41:688-696.
  10. Gaur U, Aggarwal BB (2003) Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 66:1403-1408. https://doi.org/10.1016/S0006-2952(03)00490-8
  11. Gregoire FM, Smas CM, Sul HS (1998) Understanding adipocyte differentiation. Physiol Rev 78:783-809. https://doi.org/10.1152/physrev.1998.78.3.783
  12. Grun F, Watanabe H, Zamanian Z, Maeda L, Arima K, Cubacha R, Gardiner DM, Kanno J, Iguchi T, Blumberg B (2006) Endocrine-disrupting organotin compounds are potent inducers of adipogenesis in vertebrates. Mol Endocrinol 20:2141-2155. https://doi.org/10.1210/me.2005-0367
  13. Grundler W, Dirscherl P, Beisker W, Marx K, Stampfl A, Maier K, Zimmermann I, Nusse M (2001) Early functional apoptotic responses of thymocytes induced by tri-n-butyltin. Cytometry 44:45-56. https://doi.org/10.1002/1097-0320(20010501)44:1<45::AID-CYTO1081>3.0.CO;2-Y
  14. Hare KJ, Jenkinson EJ, Anderson G (2000) An essential role for the IL-7 receptor during intrathymic expansion of the positively selected neonatal T cell repertoire. J Immunol 165:2410-2414. https://doi.org/10.4049/jimmunol.165.5.2410
  15. Ibrahimi A, Sfeir Z, Magharaie H, Amri E, Grimaldi P, Abumrad NA (1996) Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport. Proc Natl Acad Sci USA 93:2646-2651. https://doi.org/10.1073/pnas.93.7.2646
  16. Kanayama T, Kobayashi N, Mamiya S, Nakanishi T, Nishikawa J (2005) Organotin compounds promote adipocyte differentiation as agonists of the peroxisome proliferator-activated receptor gamma/retinoid X receptor pathway. Mol Pharmacol 67:766-774.
  17. Ken Nakano, Masashi Tsunoda, Nobuhiro Konno (2004) Tributyltin (TBT) increases TNF$\alpha$ mRNA expression and induces apoptosis in the murine macrophage cell line in vitro. Environ Health Prev Med 9:266-271. https://doi.org/10.1007/BF02898141
  18. Kim SK, Kim JH, Han JH, Yoon YD (2008) Inhibitory effect of tributyltin on expression of steroidogenic enzymes in mouse testis. Int J Toxicol 27:175-182. https://doi.org/10.1080/10915810801977906
  19. Kirchner S, Kieu T, Chow C, Casey S, Blumberg B (2010) Prenatal exposure to the environmental obesogen tributyltin predisposes multipotent stem cells to become adipocytes. Mol Endocrinol 24:526-539. https://doi.org/10.1210/me.2009-0261
  20. Le Maire A, Grimaldi M, Roecklin D, Dagnino S, Vivat-Hannah V, Balaguer P, Bourguet W (2009) Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Rep 10:367-373. https://doi.org/10.1038/embor.2009.8
  21. Li X, Ycaza J, Blumberg B (2011) The environmental obesogen tributyltin chloride acts via peroxisome proliferator activated receptor gamma to induce adipogenesis in murine 3T3-L1 preadipocytes. J Steroid Biochem Mol Biol 127:9-15. https://doi.org/10.1016/j.jsbmb.2011.03.012
  22. Linton PJ, Dorshkind K (2004) Age-related changes in lymphocyte development and function. Nat Immunol 5:133-139.
  23. Liston A, Lesage S, Wilson J, Peltonen L, Goodnow CC (2003) Aire regulates negative selection of organ-specific T cells. Nat Immunol 4:350-354. https://doi.org/10.1038/ni906
  24. Liu Y, Wang L, Kikuiri T, Akiyama K, Chen C, Xu X, Yang R, Chen W, Wang S, Shi S (2011) Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-$\alpha$. Nat Med 17:1594-1601. https://doi.org/10.1038/nm.2542
  25. Love PE, Bhandoola A (2011) Signal integration and crosstalk during thymocyte migration and emigration. Nat Rev Immunol 11:469-477. https://doi.org/10.1038/nri2989
  26. Lynch HE, Goldberg GL, Chidgey A, Van den Brink MR, Boyd R, Sempowski GD (2009) Thymic involution and immune reconstitution. Trends Immunol 30:366-373. https://doi.org/10.1016/j.it.2009.04.003
  27. Mandrup S, Lane MD (1997) Regulating adipogenesis. J Biol Chem 272:5367-5370. https://doi.org/10.1074/jbc.272.9.5367
  28. Maraskovsky E, O'Reilly LA, Teepe M, Corcoran LM, Peschon JJ, Strasser A (1997) Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice. Cell 89:1101-1109. https://doi.org/10.1016/S0092-8674(00)80297-4
  29. Maria G, Francesca S, Maddalena P, Simona P, Barbara M, Margherita M, Tambet T, Giacomo CG, Fabio G (1998) Epithelial V-like antigen (EVA), a novel member of the immunoglobulin superfamily, expressed in embryonic epithelia with a potential role as homotypic adhesion molecule in thymus histogenesis. J Cell Biol 141:1061-1071. https://doi.org/10.1083/jcb.141.4.1061
  30. Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass CK, Gonzalez FJ, Grimaldi PA, Kadowaki T, Lazar MA, O'Rahilly S, Palmer CN, Plutzky J, Reddy JK, Spiegelman BM, Staels B, Wahli W (2006) International Union of Pharmacology. LXI. Peroxisome proliferator- activated receptors. Pharmacol Rev 58:726-741. https://doi.org/10.1124/pr.58.4.5
  31. Micheau O, Tschopp J (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114:181-190. https://doi.org/10.1016/S0092-8674(03)00521-X
  32. Middlebrook AJ, Lebsack T, DeLuca D (2007) TNF-alpha mediated modulation of T cell development and exacerbation of in vitro T1DM in fetal thymus organ culture. J Autoimmun 29:134-145. https://doi.org/10.1016/j.jaut.2007.06.002
  33. Murray J, Barbara JA, Dunkley SA, Lopez AF, Van Ostade X, Condliffe AM, Dransfield I, Haslett C, Chilvers ER (1997) Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro. Blood 90:2772-2783.
  34. Nakanishi T (2008) Endocrine disruption induced by organotin compounds; organotins function as a powerful agonist for nuclear receptors rather than an aromatase inhibitor. J Toxicol Sci 33:269-276. https://doi.org/10.2131/jts.33.269
  35. Raffray M, Cohen GM (1993) Thymocyte apoptosis as a mechanism for tributyltin-induced thymic atrophy in vivo. Arch Toxicol 67:231-236. https://doi.org/10.1007/BF01974341
  36. Shao D, Lazar MA (1997) Peroxisome proliferator activated receptorγ, CCAAT/ enhancer-binding protein $\alpha$, and cell cycle status regulate the commitment to adipocyte differentiation. J Biol Chem 272:21473-21478. https://doi.org/10.1074/jbc.272.34.21473
  37. Shawky S, Emons H (1998) Distribution pattern of organotin compounds at different trophic levels of aquatic ecosystems. Chemosphere 36:523-535. https://doi.org/10.1016/S0045-6535(97)10011-X
  38. Smialowicz RJ, Riddle MM, Rogers RR, Luebke RW, Copeland CB (1989) Immunotoxicity of tributyltin oxide in rats exposed as adults or pre-weanlings. Toxicology 57:97-111. https://doi.org/10.1016/0300-483X(89)90037-1
  39. Snoeij NJ, Penninks AH, Seinen W (1988) Dibutyltin and tributyltin compounds induce thymus atrophy in rats due to a selective action on thymic lyphoblasts. Int J Immunopharmacol 10:891-899. https://doi.org/10.1016/0192-0561(88)90014-8
  40. Song YH, Joung JE, Lee HA, Hong JH, Yang H (2010) Tributyltin increase adipogenesis and apoptosis of rat testicular interstitial cells. Dev Reprod 4:297-306.
  41. Taub DD, Longo DL (2005) Insights into thymic aging and regeneration. Immunol Rev 205:72-93. https://doi.org/10.1111/j.0105-2896.2005.00275.x
  42. Tomiyama K, Yamaguchi A, Kuriyama T, Arakawa Y (2009) Analysis of mechanisms of cell death of Tlymphocytes induced by organotin agents. J Immunotoxicol 6:184-193. https://doi.org/10.1080/15476910903100066
  43. Trigueros C, Hozumi K, Silva-Santos B, Bruno L, Hayday AC, Owen MJ, Pennington DJ (2003) Pre-TCR signaling regulates IL-7 receptor alphaexpression promoting thymocyte survival at the transition from the doublenegative to double-positive stage. Eur J Immunol 33:1968-1977. https://doi.org/10.1002/eji.200323831
  44. Ueno S, Kashimoto T, Susa N, Asai T, Kawaguchi S, Takeda-Homma S, Terada Y, Sugiyama M (2009) Reduction in peripheral lymphocytes and thymus atrophy induced by organotin compounds in vivo. J Vet Med Sci 71:1041-1048. https://doi.org/10.1292/jvms.71.1041
  45. Yang H, Youm YH, Vishwa DD (2009) Inhibition of thymic adipogenesis by caloric restriction is coupled with reduction in age-related thymic involution. J Immunol 183:3040-3052. https://doi.org/10.4049/jimmunol.0900562
  46. Youm YH, Yang H, Amin R, Smith SR, Leff T, Dixit VD (2010) Thiazolidinedione treatment and constitutive- PPARgamma activation induces ectopic adipogenesis and promotes age-related thymic involution. Aging Cell 9:478-489. https://doi.org/10.1111/j.1474-9726.2010.00574.x
  47. Zucker RM, Elstein KH, Thomas DJ, Rogers JM (1994) Tributyltin and dexamethasone induce apoptosis in rat thymocytes by mutually antagonistic mechanisms. Toxicol Appl Pharmacol 127:163-170. https://doi.org/10.1006/taap.1994.1150