Weaver S A, Russo M P, Wright K L, Kolios G, Jobin C, Robertson D A, Ward S G
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, England.
Gastroenterology. 2001 Apr;120(5):1117-27. doi: 10.1053/gast.2001.23257.
Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells.
In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography.
The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly.
COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.
环氧化酶（COX）-2在大多数结肠癌及炎症性肠病中上调，其中肿瘤坏死因子（TNF）-α被认为起核心作用。最近有人推测TNF-α可激活磷脂酰肌醇3激酶（PI 3激酶）及其在调控由核因子κB（NF-κB）控制的基因中的作用。我们研究了PI 3激酶对结肠上皮细胞中COX-2表达的调控作用。
在HT-29和Caco-2结肠上皮细胞中，通过Northern和Western分析观察到TNF-α或白细胞介素（IL）-1α可诱导COX-2表达。通过酶联免疫吸附测定法测量前列腺素E2（PGE2）生成量来评估COX-2活性。通过电泳迁移率变动分析评估NF-κB结合活性。通过高效液相色谱法定量PI 3激酶依赖性D-3脂质产物的积累来测量PI 3激酶活性。
PI 3激酶抑制剂渥曼青霉素在HT-29和Caco-2细胞中均以浓度依赖性方式上调诱导的COX-2表达。另一种PI 3激酶抑制剂LY294002在浓度≤1μmol/L时可使HT-29细胞中诱导的COX-2信使核糖核酸（mRNA）上调。已知可激活PI 3激酶的IL-4和IL-13可下调HT-29细胞中COX-2的mRNA、蛋白质及PGE2生成。在HT-29细胞中，PI 3激酶抑制未改变NF-κB结合活性，其中TNF-α可直接激活PI 3激酶。
COX-2受PI 3激酶负调控；我们提出IL-4和IL-13的抑制作用是通过PI 3激酶依赖性途径介导的。该机制似乎不涉及NF-κB，因为PI 3激酶抑制未改变NF-κB结合活性。TNF-α除诱导COX-2外还可直接激活PI 3激酶。
