IL-1α-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCζ-ROS pathway
IL-1α-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCζ-ROS pathway
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DOI：
10.1016/s0016-5085(03)00399-8
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发表时间：
2003-06-01
期刊：
影响因子：
29.4
通讯作者：
Powell, DW
中科院分区：
文献类型：
--
作者：
Di Mari, JF;Mifflin, RC;Powell, DW
背景与目的：肠肌成纤维细胞（IMFs）在息肉发展早期表达环氧化酶2 (COX-2)，并对促炎细胞因子作出反应。白细胞介素（il -1 α）通过丝裂原活化蛋白激酶（MAPK）、蛋白激酶C （PKC）和核因子κ B （nf - κ B）依赖途径诱导COX-2在IMF中的表达。由于NF-kappaB活性可以通过PKC激活和活性氧（ROS）产生介导，因此我们研究了这些途径与il -1 α诱导的COX-2表达的关系。方法：研究特异性PKC抑制剂和抗氧化剂对PKC活化、ROS生成和COX-2表达的影响。结果：免疫沉淀/激酶（IPK）分析显示，IL-1alpha在5分钟内分别使PKC ot、8和活性增加4.5倍、3.1倍和2.6倍。2'，7-二氯荧光素（DCF）负载细胞的单细胞荧光显微镜显示，il -1 α在15分钟内使ROS水平增加2倍，这种增加被10 μ mol/L双吲哚-马来酰亚胺I （BIS）抑制，BIS是一种泛特异性PKC抑制剂，也抑制COX-2的表达。氯化车车草碱（CC） （0.5 μ mol/L）抑制经典和新型PKC活性，但不抑制PKCxi活性，并使il -1 α介导的ROS生成增强4.0倍，COX-2表达增强1.8倍。PKC假底物的使用阻止了IL-1增加ROS高于对照水平，并消除了IL-1 α诱导的COX-2表达。PKCt的小抑制RNA （slRNA）证实了其在COX-2表达中的作用。抗氧化剂抑制ROS的产生，并减少il -1 α诱导的COX-2表达80%，而不影响PKC的激活。报告基因分析表明，PKC抑制剂和抗氧化剂都不能阻止nf - kappab介导的转录。结论：PKCt和阈值ROS的产生对il -1 α诱导的COX-2表达至关重要，并与IMF中NF-kappaB易位一起起作用。
Background & Aims: Intestinal myofibroblasts (IMFs) express cyclooxygenase 2 (COX-2) early on in polyp progression and respond to pro-inflammatory cytokines. Interleukin (IL-1alpha induces COX-2 expression in IMF via mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and nuclear factor kappa B (NF-kappaB)-dependent pathways. Because NF-kappaB activity can be mediated by PKC activation and reactive oxygen species (ROS) generation, we examined the relationship of these pathways to IL-1alpha-induced COX-2 expression. Methods: The effects of specific PKC inhibitors and antioxidants on PKC activation, ROS generation, and COX-2 expression were studied. Results: Immunoprecipitation/kinase (IPK) analysis showed that IL-1alpha increased PKC ot, 8, and activity 4.5-, 3.1-, and 2.6-fold, respectively, within 5 minutes. Single-cell fluorescence microscopy of 2',7-dichlorofluorescin diacetate (DCF)-loaded cells showed that IL-1alpha increased ROS levels 2-fold within 15 minutes and this increase was inhibited by 10 mumol/L bisindoly-lymaleimide I (BIS), a pan-specific PKC inhibitor that also inhibits COX-2 expression. Chelerythrine chloride (CC) (0.5 mumol/L) inhibited classic and novel PKC activity, but not PKCxi, and enhanced IL-1alpha-mediated ROS generation 4.0-fold and COX-2 expression 1.8-fold. The use of a PKC pseudosubstrate prevented IL-1 from increasing ROS greater than control levels and abolished IL-1alpha-induced COX-2 expression. Small inhibitory RNA (slRNA) for PKCt confirmed its role in COX-2 expression. Antioxidants inhibited ROS generation and diminished IL-1alpha-induced COX-2 expression by 80%, without affecting PKC activation. Neither the PKC inhibitors nor the antioxidants prevented NF-kappaB-mediated transcription as determined by reporter gene analysis. Conclusions: PKCt and threshold ROS generation are critical for IL-1alpha-induced COX-2 expression and act concomitantly with NF-kappaB translocation in IMF.