Identification of Mouse CYP2C Involved in Arachidonic Acid

目的：关于CYP2Cs的内源性功能知之甚少。我们假设这些酶在生理上重要的内源性底物如花生四烯酸和维甲酸（维生素A）的代谢中起重要作用。我们的目标是确定小鼠中所有CYP2Cs，确定它们对特定类二十烷代谢产物产生的贡献，并确定它们在这些组织中的生理作用。已知p450衍生的AA代谢物在许多组织中具有强大的生物作用。这些效应取决于产物的区域化学和立体化学。我们正试图确定这些同种异构体的器官和细胞特异性定位。一个长期的目标是产生基因敲除小鼠来研究这类酶的生理重要性，并在细胞系中过表达这些酶以进一步确定它们的功能。这些研究以小鼠为模型来确定CYP2Cs的内源性功能。我们实验室最近克隆了5个小鼠CYP2C亚家族成员（CYP2C29、CYP2C37、CYP2C38、CYP2C39和CYP2C40），在大肠杆菌中表达了重组P450蛋白，并表明它们在花生四烯酸代谢中具有活性。我们使用Western blots、PCR和克隆技术以及免疫组织化学来确定这些亚型的器官和细胞特异性定位。Western blotting和RT-PCR显示，肺中CYP2C29含量较高。CYP2C40在结肠、盲肠、肠道、肾脏和心脏中含量较高。CYP2C40将花生四烯酸（AA）代谢为16R-和16S-HETE。这是发现的第一个将16-HETE作为主要产品的P450。肠微粒体也产生16-HETE。16R-HETE被认为在肾脏血管扩张和抑制中性粒细胞聚集和粘附等过程中很重要。利用通用引物，对产物进行亚克隆和测序，鉴定出结肠和盲肠中的2C mrna仅含有CYP2C40，肾脏中含有CYP2C40和一个新的cyp2c成员。肺主要含CYP2C29，部分含CYP2C40。血管内皮细胞中存在CYP2C29，少量存在CYP2C40。Western blot和RT-PCR提示肺组织中含有大量CYP2C29。动脉内皮细胞中含有CYP2C29和部分CYP2C40。这些研究表明CYP2Cs可能是心脏、肠道、结肠、内皮细胞和肺中重要的生理酶。

AIMS:Little is known about the endogenous function of the CYP2Cs. We postulate that these enzymes have important roles in the metabolism of physiologically important endogenous substrates such as arachidonic acid and retinoic acid (Vitamin A). Our goal has been to identify all CYP2Cs in the mouse, determine their contribution to the production of particular eicosanoid metabolites and to identify their physiological role in these tissues. The P450-derived AA metabolites are known to possess potent biological actions in numerous tissues. These effects depend on the regio- and stereochemistry of the products. We are attempting to determine the organ and cell-specific localization of these isoforms. A long-range goal is to produce knockout mice to study the physiological importance of this class of enzymes, and to overexpress these enzymes in cell lines to further establish their function.ACCOMPLISHMENTS These studies were initiated to determine the endogenous function of the CYP2Cs using the mouse as a model. Our laboratory recently cloned five members of the mouse CYP2C subfamily (CYP2C29, CYP2C37, CYP2C38, CYP2C39 and CYP2C40), expressed the recombinant P450 proteins in E. coli, and showed that they are active in the metabolism of arachidonic acid. We determined the organ and cell-specific localization of these isoforms using Western blots, PCR and cloning techniques and immunohistochemistry. Western blotting indicated and RT-PCR indicted that lung contained relatively large amounts of CYP2C29, . CYP2C40 was found in relatively high amounts in colon, cecum, gut, in kidney and heart. CYP2C40 metabolized arachidonic acid (AA) to 16R- and 16S-HETE . This is the first P450 found to produce 16-HETE as a primary product. Intestinal microsomes also produced 16-HETE. 16R-HETE is thought to be important in processes such as renal vasodilation and inhibiting neutrophil aggregation and adhesion. Using universal primers and subcloning and sequencing the products, the 2C mRNAs in colon and cecum were identified as solely CYP2C40, kidney contained CYP2C40 and a new unidentified member of the CYP2Cs. Lung contained mainly CYP2C29 and some CYP2C40. CYP2C29 was found in endothelial cells lining blood vessels, with some CYP2C40. Western blotting indicated and RT-PCR indicted that lung contained large amounts of CYP2C29. Endothelial cells of arteries contained CYP2C29 and some CYP2C40.These studies indicate that CYP2Cs may be important physiological enzymes in heart, gut, colon, endothelial cells, and lung.