Williams Shalmica R, Son Deok-Soo, Terranova Paul F
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
Toxicology. 2004 Jan 15;195(1):1-17. doi: 10.1016/j.tox.2003.08.005.
Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24h of treatment, and pre-treatment with (1 microM) of either a general PKC inhibitor (BisI) or PKCdelta-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCalpha, beta, epsilon, tau, lambda and RACK1. PKCgamma, eta, theta and DGKtheta were not detected. TCDD (1 nM) increased PKCdelta protein approximately eight-fold after 24h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCdelta, from the cytoplasm and the nucleus to the perinuclear area after 2h of treatment, however, after 24h of treatment PKCdelta was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCdelta-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCdelta-specific activities increased after 1-2h of treatment. Then TCDD increased the total PKC activity again after 12h of treatment, whereas, PKCdelta-specific activity resurged at 24h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCdelta protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCdelta as an effector molecule for TCDD-mediated biological events in this ovarian cancer cell line.
2,3,7,8-四氯二苯并-对-二恶英（TCDD）与蛋白激酶C（PKC）信号通路之间的相互作用以细胞和组织特异性方式调控，尽管其生理意义尚不清楚。本研究旨在利用小鼠卵巢表面上皮癌细胞系（ID8）确定TCDD对PKC通路的影响。佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）在处理24小时后增强了（1 nM）TCDD诱导的7-乙氧基异吩恶唑酮-O-脱乙基酶（EROD）活性，用（1 microM）的通用PKC抑制剂（BisI）或PKCδ特异性抑制剂（Rotterlin）预处理可消除这种增强作用，表明PKC的激活增强了TCDD信号转导。蛋白质印迹分析显示，未刺激的ID8细胞表达PKCα、β、ε、τ、λ和RACK1。未检测到PKCγ、η、θ和DGKθ。TCDD（1 nM）在处理24小时后使PKCδ蛋白增加约8倍，且这种效应呈剂量依赖性（0.1 - 100 nM）；所检测的其他PKC同工型和相关信号蛋白不受TCDD处理的影响。免疫荧光显微镜检查显示，TCDD（1 nM）在处理2小时后促进了PKCδ的亚细胞重新分布，从细胞质和细胞核转移至核周区域，然而，在处理24小时后，在类似核仁的核结构中观察到PKCδ。TCDD（1 nM）还以双相时间响应方式增加了总PKC和PKCδ特异性激酶活性。处理1 - 2小时后总PKC和PKCδ特异性活性增加。然后TCDD在处理12小时后再次增加总PKC活性，而PKCδ特异性活性在处理24小时时恢复并在处理后48小时保持升高。结果表明，TCDD优先诱导PKCδ蛋白表达和磷酸转移酶活性及其膜转位，表明在该卵巢癌细胞系中PKCδ作为TCDD介导的生物学事件的效应分子具有潜在细胞内作用。
