Epidermal stem cell properties in mice with altered polyamines

多胺改变的小鼠表皮干细胞特性

• 批准号: 7752670
• 负责人: LISA M SHANTZ
• 金额: $ 7.76万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7752670
• 关键词: Address; Anabolism; Back; Binding; Biological Models; Bromodeoxyuridine; CD34 gene; Carcinogens; Cell Cycle Regulation; Cell Proliferation; Cell Separation; Cells; Chemopreventive Agent; Chronic; Cyclin D1; DL-alpha-Difluoromethylornithine; Development; Differentiation Antigens; Down-Regulation; Enzymes; Epidermis; Event; Exposure to; Future; Genes; Ha-ras Oncogene; Hair follicle structure; Human; Hyperplasia; In Vitro; Integrins; Keratin; Label; Lead; MEKs; Malignant Neoplasms; Measures; Modeling; Molecular; Monitor; Mus; Mutation; Nude Mice; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitor; Pathway interactions; Phenotype; Polyamines; Population; Prevention; Property; Proteins; Public Health; Role; Signal Transduction Pathway; Skin; Skin Cancer; Skin Carcinogenesis; Skin Carcinoma; Skin Neoplasms; Skin Transplantation; Skin graft; Small Interfering RNA; Sorting - Cell Movement; Stem cells; Stratum Basale; Study models; Techniques; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Tumor Promotion; Tumor Suppressor Proteins; Undifferentiated; Up-Regulation; Wild Type Mouse; Work; base; cell growth; cell type; in vivo; in vivo Model; inhibitor/antagonist; interest; keratin 5; keratinocyte; keratinocyte differentiation; knock-down; mouse model; novel; overexpression; promoter; research study; response; self-renewal; skin cancer prevention; stem; three-dimensional modeling; transgene expression; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Although it is known that the mammalian epidermis is maintained by self-renewal of stem cells, the underlying mechanisms, and the changes that occur in skin cancer, are not well-defined. A more thorough understanding of stem cell proliferation and differentiation will provide potential targets for skin cancer prevention. One of the most widely used models for the study of skin carcinogenesis is the mouse model of initiation and promotion. The initiating event occurs when exposure to a carcinogen induces genetic changes in epidermal stem cells, which are thought to be located in the bulge region of the hair follicle. Initiation causes activation of the Ha-ras oncogene and chronic up-regulation of the Raf/MEK/ERK pathway. Our previous work demonstrates that the polyamine metabolizing enzyme ornithine decarboxylase (ODC) is downstream of MEK, and induction of ODC activity is necessary for skin tumor development. We have also shown that antizyme (AZ), which binds to ODC and targets it for degradation, is a potent tumor suppressor in skin carcinogenesis. The central hypothesis of the proposed work is that the tumor suppressor effects of AZ are associated with decreased proliferation of epidermal stem cells and induction of keratinocyte differentiation. The proposed experiments use mice with expression of AZ in the skin, along with either MEK or cyclin D1 (CD1), which like ODC is a downstream target of MEK. Expression of each transgene is driven by either the Keratin 5 or Keratin 14 promoter, thus directing expression primarily to the hair follicle bulge and modifying important signal transduction pathways in the population of cells crucial to stem cell expansion, keratinocyte differentiation and tumorigenesis. Preliminary studies with mice expressing both AZ and CD1 have led to the hypothesis that reduced ODC activity resulting from targeted AZ expression restricts cell growth in epidermal stem cells and inhibits tumorigenesis at least in part by suppressing CD1 activity. Aim 1 will measure stem cell expansion in bulge cells of MEK, MEK/AZ, CD1 and CD1/AZ mice using BrdU labeling and expression of a6-integrin and CD34, both markers of epidermal stem cells. We will directly address whether CD1 is controlled by ODC in bulge cells by isolating these cells from several transgenic lines using FACS sorting and monitoring CD1 expression. Aim 2 will use organotypic raft cultures of human keratinocytes expressing MEK, CD1 and/or AZ. This three-dimensional model system will allow us to address changes in tissue organization and expression of differentiation markers in response to transgene expression. The possibility that AZ is acting independently of ODC will be addressed by comparing results in AZ-expressing cells to those in cells expressing siRNA targeted to the ODC protein, thus knocking down ODC without changing AZ expression. To extend these studies to an in vivo model, human keratinocytes expressing our genes of interest will be grafted onto athymic mice, and changes in epidermal proliferation and differentiation will be measured. Information obtained from the proposed work will lead to future studies that address the broader question of what pathways are altered in follicular stem cells upon ODC downregulation.

描述(由申请人提供)： 虽然已经知道哺乳动物的表皮是通过干细胞的自我更新来维持的，但皮肤癌的潜在机制和发生的变化还不是很清楚。更深入地了解干细胞的增殖和分化将为皮肤癌的预防提供潜在的靶点。研究皮肤癌发生的最广泛使用的模型之一是小鼠的启动和促进模型。当暴露在致癌物中导致表皮干细胞发生基因变化时，就会发生这种启动事件，表皮干细胞被认为位于毛囊的隆起区域。启动导致Ha-ras癌基因的激活和Raf/MEK/ERK通路的慢性上调。我们以前的工作表明，多胺代谢酶鸟氨酸脱羧酶(ODC)位于MEK的下游，诱导ODC活性对皮肤肿瘤的发生是必要的。我们还表明，抗酶(AZ)与ODC结合并针对其进行降解，在皮肤癌的发生中是一种有效的肿瘤抑制因子。这项工作的中心假设是AZ的肿瘤抑制作用与减少表皮干细胞的增殖和诱导角质形成细胞分化有关。拟议的实验使用了皮肤中表达AZ的小鼠，以及MEK或细胞周期蛋白D1(CD1)，后者和ODC一样是MEK的下游靶点。每一种转基因的表达都是由角蛋白5或角蛋白14启动子驱动的，从而将表达主要导向毛囊隆起，并修改对干细胞扩张、角质形成细胞分化和肿瘤发生至关重要的细胞群体中的重要信号转导途径。对同时表达AZ和CD1的小鼠的初步研究导致了一种假设，即靶向AZ表达导致的ODC活性降低会限制表皮干细胞中的细胞生长，并至少部分通过抑制CD1活性来抑制肿瘤的发生。目的1通过BrdU标记和表皮干细胞标志物α6整合素和CD34的表达，检测MEK、MEK/AZ、CD1和CD1/AZ小鼠膨大细胞中干细胞的扩增情况。我们将通过从几个转基因细胞系中分离出这些细胞，使用FACS分类和监测CD1的表达，直接解决CD1是否受ODC控制的问题。目的2将使用器官分型移植物培养表达MEK、CD1和/或AZ的人角质形成细胞。这个三维模型系统将使我们能够处理组织结构和分化标记表达的变化，以响应转基因表达。AZ独立于ODC发挥作用的可能性将通过比较表达AZ的细胞和表达针对ODC蛋白的siRNA的细胞中的结果来解决，从而在不改变AZ表达的情况下击倒ODC。为了将这些研究扩展到体内模型，表达我们感兴趣的基因的人角质形成细胞将被移植到无菌小鼠身上，并将测量表皮增殖和分化的变化。从拟议的工作中获得的信息将导致未来的研究，以解决更广泛的问题，即卵泡干细胞在ODC下调调控时改变了哪些途径。