KERATIN FUNCTION IN EPIDERMAL GROWTH AND DIFFERENTIATION

表皮生长和分化中的角蛋白功能

• 批准号: 3457529
• 负责人: Kathryn Marie Albers
• 金额: $ 8.17万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3457529
• 关键词: SDS polyacrylamide gel electrophoresis; cell differentiation; cell growth regulation; cell line; cytology; electron microscopy; epithelium; gene deletion mutation; gene expression; genetically modified animals; human genetic material tag; immunofluorescence technique; immunologic assay /test; intercellular connection; intermediate filaments; keratin; laboratory mouse; northern blottings; protein biosynthesis; protein engineering; protein structure function; skin; southern blotting; tissue /cell culture; transcription factor; transfection; western blottings

Expression of keratin intermediate filament proteins is highly regulated during the development, growth, and differentiation of the epidermis. Our understanding of the function of these proteins and the network they assemble into is limited, though their role in the cell seems to be one other than purely structural. The broad, long-term objective of this proposal is to determine the functional significance of the keratins within epithelial cells and the role they have in the growth and differentiation of the epidermis. The Specific Aims are 1) to determine how the expression of mutant keratin proteins in cultured epithelial cells affects cell growth, expression of cell differentiation, cell morphology, regulation of keratin synthesis and filament assembly, and 2) To determine how mutant keratin proteins expressed in the epidermis of transgenic mice affect in vivo epidermal growth parameters, expression of differentiation, and morphology of the epidermis. Both cultured cells and transgenic mouse model systems will be employed to create a comprehensive system that incorporates both flexibility and relevancy. Permanent cell lines will be created by stably transfecting epithelial cells with previously isolated deletion mutations of a human K14 epidermal keratin cDNA. Mutant K14 cDNAs will be expressed in cells using the dexamethasone inducible MMTV LTR promoter in order to achieve control of mutant expression and to isolate lines that express mutant keratins that may be lethal. The inducible expression of mutant keratins in cultured cells will enable study of the cellular response to an abnormal keratin network with respect to cell growth, differentiation, morphology, keratin synthesis and filament assembly. Expression of mutant K14 keratin in transgenic mouse epidermis will allow a direct assessment of the effect an abnormal keratin network has on the structure and integrity of the epidermis and on parameters of epidermal growth and differentiation. Keratin filament structure and organization are known to be altered in certain genetic diseases of the epidermis, thus, the expression of generically defective keratins which produce abnormal keratin networks in the transgenic mouse epidermis will provide a powerful system in which to evaluate the role of keratin networks in the function and structure of the epidermis. The effect of mutant keratin expression on cell growth will be determined by measuring cell doubling time and labeling indices. Whether mutant keratin expression affects the onset and progression of epidermal cell differentiation will be determined by immunolabeling and PAGE to detect differentiation specific proteins, counting cornified envelopes, and morphology. Morphology of cells expressing mutant keratin will be evaluated using light and electron microscopy in conjunction with immunolabeling. Previous studies suggest that expression of mutant keratin proteins inhibits keratin synthesis. To investigate this possibility, keratin synthesis will be measured on the transcriptional and translational levels in cans expressing abnormal keratin using Northern analysis and one- and two-dimensional PAGE.

角蛋白中间丝蛋白的表达受到高度调节 在表皮的发育、生长和分化过程中。 我们 了解这些蛋白质的功能及其网络， 虽然它们在细胞中的作用似乎是一体的， 除了纯粹的结构性。 这一广泛而长期的目标 建议是确定角蛋白的功能意义， 上皮细胞及其在生长和分化中的作用 的表皮。 具体目标是：1）确定表达方式 在培养的上皮细胞中的突变角蛋白影响细胞 生长，细胞分化表达，细胞形态， 角蛋白合成和细丝组装，和2）为了确定如何突变 角蛋白在转基因小鼠表皮中的表达影响 体内表皮生长参数，分化表达，和 表皮的形态。 培养细胞和转基因小鼠 模型系统将被用来创建一个全面的系统， 既有灵活性，又有相关性。 永久细胞系将是 通过稳定地分离上皮细胞与先前分离的 人K14表皮角蛋白cDNA的缺失突变。 突变型K14 cDNA 将使用地塞米松诱导的MMTV LTR在细胞中表达 启动子，以实现突变体表达的控制，并分离 表达突变角蛋白的细胞系可能是致命的 诱导型 突变角蛋白在培养细胞中的表达将使研究 细胞对异常角蛋白网络的反应 生长、分化、形态、角蛋白合成和纤维 组装件. 突变型K14角蛋白在转基因小鼠表皮中的表达 将允许对异常角蛋白网络的影响进行直接评估 对表皮的结构和完整性以及 表皮生长和分化。 角蛋白丝结构和 已知在某些遗传疾病中， 表皮，因此，表达一般缺陷角蛋白， 在转基因小鼠表皮中产生异常的角蛋白网络， 提供了一个强大的系统，以评估角蛋白网络的作用， 表皮的功能和结构。 突变体效应 角蛋白对细胞生长的表达将通过测量细胞中的角蛋白含量来确定。 倍增时间和标记指数。 突变角蛋白表达 影响表皮细胞分化的发生和进展， 通过免疫标记和PAGE测定以检测分化特异性 蛋白质、计数皮质包膜和形态学。 形态 表达突变角蛋白的细胞将使用光和电子技术进行评估。 显微镜检查结合免疫标记。 以往的研究表明 突变角蛋白的表达会抑制角蛋白的合成。 到 研究这种可能性，角蛋白的合成将在 在表达异常的Can中的转录和翻译水平 使用北方分析和一维和二维PAGE分析角蛋白。