2,3,7,8-tetrachlorodibenzo-p-dioxin에 의한 세뇨관 상피세포에서의 Fibronectin 분비에 미치는 활성산소족과 Mitogen-activated Protein Kinase의 역할 = Role of Reactive Oxygen Species and Mitogen-activated Protein Kinases in 2,3,7,8-tetrachlorodibinzo-p-dioxin-induced Fibronectin Secretion by MDCK Cells

다국어 초록 (Multilingual Abstract)

Background:2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD:dioxin) is a potent environmental toxicant that alters various cell function. Both reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) mediate dioxin-induced cytotoxicity. Since dioxin was shown to increase renal cell fibronectin secretion in a dose-dependent manner and ROS and MAPK also play roles in fibronectin upregulation in renal cells, the present study examined whether ROS and/or MAPK activation play a role in dioxin-induced fibronectin upregulation in tubular epithelial cells.
Methods:Madin-Darby canine kidney (MDCK) cells were cultured with minimum essential medium (MEM) containing 10% fetal bovine serum. Growth arrested and synchronized MDCK cells by serum deprivation were stimulated with dioxin 1 nM in the presence or absence of extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 50 µM, p38 MAPK inhibitor 100 nM, trolox 500 µM, or taurine 500 µM for up to 48 hours. Dichlorofulorescein (DCF)-sensitive cellular ROS was measured by FACScan and fibronectin in the media and cellular MAPK by a Western blot analysis.
Results:Dioxin 1 nM significantly increased cellular ROS and fibronectin in MDCK cells. Antioxidants, trolox and taurine, effectively inhibited dioxin-induced cellular ROS and fibronectin secretion. Dioxin increased phosphorylation of ERK at 5 minutes and P38 MAPK at 48 hours. Dioxin did not affect c-Jun NH2-terminal kinase (JNK) activation for up to 48 hours. Both PD98059 and p38 MAPK inhibitor suppressed dioxin-induced fibronectin secretion by MDCK cells.
Conclusion:These data suggest that dioxin increases fibronectin secretion by renal distal tubular epithelial cells through ROS and MAPK (ERK and p38 MAPK) and this may lead to renal fibrosis. (Korean J Nephrol 2005;24(3):350-357)

국문 초록 (Abstract)

목 적:2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD:dioxin)은 신장을 비롯한 다양한 조직과 세포의 기능에 영향을 미치는 독성이 대단히 강한 내분비계 장애물질이다. 활성산소족 (reactive oxygen species, ROS)과 mitogen-activated protein kinases (MAPK)는 dioxin에 의한 세포 독성을 매개하고 신장세포에서는 다양한 자극에 의한 fibronectin 상향조절을 매개하는 것으로 알려져 있다. Dioxin은 신장세포에서 농도 의존적으로 fibronectin 분비를 상향조절하므로 본 연구는 dioxin에 의한 세뇨관 상피세포에서의 fibronectin 분비 상향조절에 ROS와 MAPK 활성화가 관여하는지를 검색하였다.
방 법:개의 원위 세뇨관 상피세포 (Madin-Darby canine kidney, MDCK)를 10% 우태아 혈청을 함유한 minimum essential medium (MEM)에서 배양하였다. 혈청 배제 MEM에 24시간 동안 배양하여 세포 성장을 동일화 시킨 후 dioxin 1 nM로 5분-48시간 동안 자극하였다. 필요에 따라 extracellular signal-regulated protein kinases (ERK) 억제제 PD98059 50 µM, p38 MAPK inhibitor [2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2- dihydropyrazol-3-one] 100 nM 또는 항산화제 trolox나 taurine 500 µM을 dioxin 투여 1시간 전부터 투여하였다. Dichlorofulorescein (DCF)에 민감한 세포내 ROS를 FACScan으로 측정하였고, 배양액으로 분비된 fibronectin과 세포내 MAPK는 Western blot 분석으로 정량하였다.
결 과:Dioxin은 MDCK 세포내 ROS를 의미있게 증가시켰고, trolox나 taurine은 dioxin에 의한 세포내 ROS 증가와 fibronectin 분비 증가를 효과적으로 억제하였다. MDCK 세포의 ERK는 dioxin 투여 후 5분에, P38 MAPK는 48시간에 의미있게 활성화되었으나 c-Jun NH2-terminal kinases (JNK) 활성화는 dioxin에 의하여 영향을 받지 않았다. PD98059나 p38 MAPK inhibitor는 dioxin에 의한 fibronectin분비 증가를 기저치까지 억제하였다.
결 론:이상의 결과는 dioxin에 의한 세뇨관 상피세포의 fibronectin 분비 증가는 ROS, ERK나 p38 MAPK에 의하여 매개됨을 시사한다.

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