Identification of Mouse CYP2C Involved in Arachidonic Acid

目的：对细胞色素P2磷酸转移酶的内源性功能知之甚少。我们推测这些酶在生理上重要的内源底物如花生四烯酸和维甲酸(维生素A)的代谢中起着重要的作用。我们的目标是确定小鼠体内所有的细胞色素P450 2 C，确定它们对特定二十烷基类代谢物产生的贡献，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学作用。这些影响取决于产品的区域和立体化学。我们正试图确定这些异构体的器官和细胞特异性定位。一个长期的目标是制造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。这些研究最初是以小鼠为模型来确定细胞周期蛋白2磷酸酶的内源性功能。本实验室最近克隆了小鼠细胞色素P450亚家族的5个成员(细胞色素P450、细胞色素P450和细胞色素P450)。我们用免疫印迹、聚合酶链式反应和克隆技术以及免疫组织化学技术确定了这些异构体的器官和细胞特异性定位。Western blotting和RT-PCR结果表明，肺组织中含有较多的细胞色素P450 2 9。CYP2C40在结肠、盲肠、肠道、肾脏和心脏中含量较高。细胞色素P450将花生四烯酸(AA)代谢成16R-和16S-HETE。这是第一个发现以16-HETE为主要产物的P450。肠道微生物体也产生了16-HETE。16R-HETE被认为在肾血管扩张、抑制中性粒细胞聚集和黏附等过程中起重要作用。利用通用引物和亚克隆测序的方法，鉴定了结肠和盲肠中的2CmRNAs为纯的CYP2C40，肾脏中含有CYP2C40和一个新的未知成员。肺中主要含有细胞色素P4502C29和少量的细胞色素P450C40。在血管衬里的内皮细胞中发现了细胞色素P4502C29，有少量的细胞色素P450C40。Western blotting和RT-PCR结果表明肺组织中含有大量的细胞色素P450 2C29。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.