Molecular biological studies on arachidonate oxygenase isozymes

12-脂氧合酶有两种同工酶，底物专一性和一级结构不同。已知的环氧合酶同工酶有两种：已知的环氧合酶(COX-1)和新近发现的环氧合酶(COX-2)。它们的生理和病理功能是积极研究的对象。本研究对病理生理条件下同工酶的诱导和同工酶基因的转录调控进行了研究，获得了以下实验结果：1)COX-1基因的转录调控：佛波酯诱导人巨核细胞CMK向巨核细胞分化。2)COX-2基因的转录调控：小鼠成骨细胞MC3T3-E1可被肿瘤坏死因子α诱导COX-2表达，NFkappaB和NF-IL6参与其中。3)12-脂氧合酶同工酶的自杀性失活：白细胞12-脂氧合酶在催化过程中明显失活，而血小板12-脂氧合酶反应几乎是线性进行的。白细胞酶的自杀性失活归因于其产生15-羟基过氧酸和12-羟基过氧酸，以及前一个次要产物转化为14，15-环氧化物。4)12-脂氧合酶基因的转录调控：与台湾12-脂氧合酶合作，在人表皮样癌A431细胞中诱导EGF，并提示SP-1参与了该酶基因的转录。此外，还观察到大鼠松果体中12-脂氧合酶mRNA的昼夜变化。

There are two isozymes of 12-lipoxygenase distinguishable in terms of substrate specificity and primary structure. Two cycloowoygenase isozymes are known : the previously known enzyme (COX-1) and the recently found isozyme (COX-2). Their physiological and pathological functions are subjects of active invesigations. This research project is for the studies on the induction of the isozymes in pathophysiological conditions and the transcriptional regulation of the isozyme genes, and the following experimental results were obtained.1) Transcriptional regulation of COX-1 gene : Addition of a phorbol ester induced COX-1 in human megakaryoblastic cells CMK along with their differentiation to megakaryocytes. We found the presence of prostaglandin D synthase in megakaryoblasts which is absent in platelets.2) Transcriptional regulation of COX-2 gene : Induction of COX-2 by TNFalpha was demonstrated with murine osteoblastic cells MC3T3-E1, and involvement of NFkappaB and NF-IL6 were shown. COX-2 induction was also observed in rat pluerisy model.3) Suicide inactivation of 12-lipoxygenase isozymes : Leukocyte 12-lipoxygenase shows a marked inactivation during its catalysis while platelet 12-lipoxygenase reaction proceeded almost linearly. The suicide inactivation of the leukocyte enzyme was attributed to its production of 15-hydroperoxy acid in addition to 12-hydroperoxy acid and to the coversion of the former minor product to a 14,15-epoxide.4) Transcriptional regulation of 12-lipoxygenase gene : In collaboration with Taiwanese group 12-lipoxygenase induction of EGF in human epidermoid carcinoma cells A431 was demonstrated, and involvement of SP-1 was suggested in the transcription of the enzyme gene. Furthermore, diurnal change of 12-lipoxygenase mRNA was observed in rat pineal gland.