MONOOXYGENASES AND KERATINOCYTE DIFFERENTIATION

单加氧酶和角质形成细胞分化

基本信息

批准号：
6193979
负责人：
DIANE S KEENEY
金额：
$ 25.34万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-08 至 2003-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6193979
关键词：
cell cell interaction cell differentiation cell growth regulation cell proliferation cytochrome P450 eicosanoid metabolism eicosanoids enzyme activity keratinocyte laboratory mouse oxygenases skin tissue /cell culture

项目摘要

DESCRIPTION: (adapted from the applicant's abstract) This application addresses the role of fatty acid metabolism in hyperproliferative skin diseases where the disease phenotype leads to abnormally high levels of eicosanoids and free fatty acids in skin lesions. While there is no unifying hypothesis explaining the roles of eicosanoids in epithelial differentiation and disease, these lipids are thought to mediate signals regulating cell proliferation and differentiation. Dr. Keeney's proposal deals with the roles of lipid mediators generated by cytochrome P450 epoxygenases in the control of epithelial cell differentiation. The applicant and colleagues have characterized keratinocyte-specific epoxygenases (CYP2B19, CYP2B12) that generate epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids from arachidonic acid. Their expression in rodent skin in epidermal keratinocyte cultures is highly differentiation-specific. Their lipid mediator (EETs) are present in normal skin, and the EETs are produced from endogenous arachidonic acid in differentiated keratinocytes. The applicant hypothesizes that P450-derived eicosanoids mediate signals regulating differentiated functions in epidermal granular cells and that ablation or overexpression of P450 epoxygenases will alter this differentiated phenotype of keratinocytes. This proposal will address 1) The endogenous CYP2B19 products that are biologically active by confirming that the EETs generated by CYP2B19 are present in murine epidermal keratinocyte cultures, coincident with CYP2B19 mRNA and protein. The applicant will prove that EETs are biologically active in murine epidermal keratinocyte cultures, coincident with mRNA and protein. 2) The applicant will demonstrate a functional relationship between endogenous EET production and the differentiated keratinocyte phenotype, and that ablation of EET production alters this phenotype. In the third specific aim, the applicant will prove that EET's are biologically active in murine epidermal keratinocyte cultures by demonstrating how the differentiation and the sensitivity of these cells to extracellular Ca2+ is altered by inappropriate overproduction of EETs. Endogenous EETs will be measured using a mass spectral assay. Chemical inhibitors and antisense cDNA will be used to ablate CYP-derived EET production. Recombinant adenoviruses expressing P450 epoxygenases (including CYP2B19) will be used to overproduce EETs intracellularly. Results of these studies will identify specific cellular pathways and granular cell functions regulated by CYP-derived eicosanoids and suggest potential mechanisms for their actions.

描述：（改编自申请人的摘要）本申请地址脂肪酸代谢在过度增殖性皮肤病中的作用疾病表型导致类二十烷酸和游离脂肪含量异常高皮肤损伤处的酸。虽然没有统一的假设来解释类二十烷酸在上皮分化和疾病中的作用，这些脂质被认为介导调节细胞增殖的信号差异化。基尼博士的提案涉及脂质介质的作用由控制上皮细胞的细胞色素 P450 环氧化酶产生差异化。申请人和同事描述了角质形成细胞特异性环氧化酶（CYP2B19、CYP2B12）产生环氧二十碳三烯酸 (EET) 和羟基二十碳四烯酸花生四烯酸。它们在啮齿动物皮肤表皮角质形成细胞中的表达文化具有高度分化特异性。他们的脂质介质（EET）是存在于正常皮肤中，EET 由内源性花生四烯酸产生分化的角质形成细胞中的酸。申请人假设 P450 衍生的类二十烷酸介导调节分化功能的信号表皮颗粒细胞和 P450 的消融或过度表达环氧化酶将改变角质形成细胞的这种分化表型。这该提案将解决 1) 生物学上的内源性 CYP2B19 产物通过确认 CYP2B19 产生的 EET 存在于小鼠体内来激活表皮角质形成细胞培养物，与 CYP2B19 mRNA 和蛋白质一致。这申请人将证明 EET 在小鼠表皮中具有生物活性角质形成细胞培养物，与 mRNA 和蛋白质一致。 2) 申请人将证明内源性 EET 产生与分化的角质形成细胞表型，以及 EET 产生的消除改变这种表型。在第三个具体目标中，申请人将证明： EET 在小鼠表皮角质形成细胞培养物中具有生物活性展示这些细胞的分化和敏感性细胞外 Ca2+ 因 EET 的不适当过量产生而改变。内源性 EET 将使用质谱分析进行测量。化学抑制剂和反义 cDNA 将用于消除 CYP 衍生的 EET 生产。表达 P450 环氧化酶的重组腺病毒（包括 CYP2B19) 将用于在细胞内过量产生 EET。这些结果研究将确定特定的细胞途径和颗粒细胞功能受 CYP 衍生的类二十烷酸调节，并提出其潜在机制行动。