Ouyang Y, Hwang S G, Han S H, Kaminski N E
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824, USA.
Mol Pharmacol. 1998 Apr;53(4):676-83. doi: 10.1124/mol.53.4.676.
2-Arachidonyl-glycerol (2-Ara-Gl) recently was identified as a putative endogenous ligand for cannabinoid receptor types CB1 and CB2 by competitive binding. More recent immune function assays demonstrated that 2-Ara-Gl possessed immunomodulatory activity. Because several plant-derived cannabinoids inhibit interleukin-2 (IL-2) expression, 2-Ara-Gl was investigated for its ability to modulate this cytokine. The direct addition of 2-Ara-Gl to mouse splenocyte cultures suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 secretion and steady state mRNA expression in a dose-dependent manner. 2-Ara-Gl also produced a marked inhibition of IL-2 promotor activity as determined by transient transfection of EL4.IL-2 cells with a pIL-2-CAT construct. 2-Ara-Gl at 5, 10, 20, and 50 microM suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 promotor activity by 18%, 28%, 39%, and 54%, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2 by 2-Ara-Gl, the DNA-binding activity of transcription factors, nuclear factor of activated T cells (NF-AT), nuclear factor for immunoglobulin kappa chain in B cells (NF-kappa B/Rel), activator protein-1(AP-1), octamer, and cAMP-response element binding protein was evaluated by electrophoretic mobility shift assay in mouse splenocytes. In addition, a reporter gene expression system for p(NF-kappa B)3-CAT, p(NF-AT)3-CAT, and p(AP-1)3-CAT was used in transiently transfected EL4.IL-2 cells to determine the effect of 2-Ara-Gl on promoter activity for each of the specific transcription factors. 2-Ara-Gl reduced both the NF-AT-binding and promoter activity in a dose-dependent manner and, to a lesser degree, NF-kappa B/Rel-binding and promoter activity. No significant effect was observed on octamer- and cAMP-response element-binding activity. AP-1 DNA-binding activity was not inhibited by 2-Ara-Gl, but a modest inhibition of promoter activity was observed.
2-花生四烯酸甘油酯（2-Ara-Gl）最近通过竞争性结合被鉴定为大麻素受体1型（CB1）和2型（CB2）的一种假定内源性配体。最近的免疫功能检测表明2-Ara-Gl具有免疫调节活性。由于几种植物源性大麻素会抑制白细胞介素-2（IL-2）的表达，因此对2-Ara-Gl调节这种细胞因子的能力进行了研究。将2-Ara-Gl直接添加到小鼠脾细胞培养物中，以剂量依赖的方式抑制了佛波醇-12-肉豆蔻酸酯-13-乙酸酯加离子霉素诱导的IL-2分泌和稳态mRNA表达。通过用pIL-2-CAT构建体瞬时转染EL4.IL-2细胞确定，2-Ara-Gl还对IL-2启动子活性产生了显著抑制。5、10、20和50微摩尔的2-Ara-Gl分别将佛波醇-12-肉豆蔻酸酯-13-乙酸酯加离子霉素诱导的IL-2启动子活性抑制了18%、28%、39%和54%。为了进一步阐明2-Ara-Gl对IL-2转录调控的机制，通过电泳迁移率变动分析法在小鼠脾细胞中评估了转录因子活化T细胞核因子（NF-AT）、B细胞中免疫球蛋白κ链核因子（NF-κB/Rel）、活化蛋白-1（AP-1）、八聚体和cAMP反应元件结合蛋白的DNA结合活性。此外，在瞬时转染的EL4.IL-2细胞中使用了p（NF-κB）3-CAT、p（NF-AT）3-CAT和p（AP-1）3-CAT的报告基因表达系统，以确定2-Ara-Gl对每种特定转录因子启动子活性的影响。2-Ara-Gl以剂量依赖的方式降低了NF-AT结合和启动子活性，并在较小程度上降低了NF-κB/Rel结合和启动子活性。未观察到对八聚体和cAMP反应元件结合活性的显著影响。AP-1的DNA结合活性未被2-Ara-Gl抑制，但观察到对启动子活性有适度抑制。
