Molecular biological studies on arachidonate oxygenase isozymes

根据底物特异性和初级结构，12-脂氧合酶有两种同工酶。已知两种环氧化酶同工酶：先前已知的酶（COX-1）和最近发现的同工酶（COX-2）。它们的生理和病理功能是积极研究的主题。本课题为病理生理条件下同工酶的诱导及同工酶基因的转录调控研究，实验结果如下：1) COX-1基因的转录调控：在人巨核母细胞CMK中加入一种酚酯诱导COX-1向巨核细胞分化。我们发现巨核细胞中存在前列腺素D合成酶，而血小板中不存在。2) COX-2基因的转录调控：小鼠成骨细胞MC3T3-E1证实TNFalpha诱导COX-2， NFkappaB和NF-IL6参与。COX-2在大鼠多发性模型中也有诱导作用。3) 12-脂氧合酶同工酶的自杀式失活：白细胞12-脂氧合酶在催化过程中表现出明显的失活，而血小板12-脂氧合酶的催化过程几乎呈线性进行。白细胞酶的自杀失活是由于它除了产生12-羟基过氧酸外还产生15-羟基过氧酸，并将前者的次要产物转化为14,15-环氧化物。4) 12-脂氧合酶基因的转录调控：与台湾研究组合作证实了12-脂氧合酶在人表皮样癌细胞A431中诱导EGF，并提示SP-1参与了该酶基因的转录。观察大鼠松果体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.