REGULATION OF HUMAN KERATINOCYTE GENE EXPRESSION BY TPA

TPA 对人类角质形成细胞基因表达的调节

• 批准号: 3162189
• 负责人: Noreen J Hickok
• 金额: $ 17.38万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3162189
• 关键词: RNA binding protein; biomarker; chimeric proteins; enzyme activity; gene deletion mutation; genetic regulation; genetic translation; human genetic material tag; immunoprecipitation; keratinocyte; messenger RNA; ornithine decarboxylase; phorbols; site directed mutagenesis; tissue /cell culture; transfection

The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) is a potent tumor promoter and causes epidermal hyperplasia in dermatological systems. In human keratinocytes, TPA induces epidermal differentiation and suppresses ornithine decarboxylase (ODC) activity without causing any concomitant changes in ODC mRNA levels. In contrast, in mouse skin, TPA is a proliferative agent, and causes increases in ODC activity, mRNA levels, and gene transcription rates; this increase in ODC activity appears to be necessary for tumor promotion, suggesting an important role for ODC in carcinogenesis. TPA thus has opposite effects in human and mouse, and the goal of these studies is to elucidate the mechanisms whereby TPA suppresses ODC gene expression in human keratinocytes. As TPA decreases ODC synthetic rates, and also appears to decrease ODC protein stability, we propose that TPA regulates ODC gene expression by regulating ODC mRNA translatability and by altering ODC enzyme half-life. The mechanisms of these effects will be studied by (1) elucidating the effects of TPA on the expression of transfected human ODC cDNA in human and mouse keratinocytes, (2) assessing the role of the ODC mRNA 51-noncoding (5'-nc) region in mediating the effect of TPA on ODC mRNA translatability by transfection of 5'-nc region deletants, (3) elucidating if the effect of TPA on ODC mRNA translatability is mediated by protein binding to the ODC mRNA 5'-nc region using RNA-protein UV cross-linking assays, (4) determining which sequences in the ODC protein mediate its destabilization by TPA, by transfection of probein-coding region deletants and determining enzyme half-life, and (5) isolating the protein(s) interacting with the ODC sequences that mediates the effect of TPA on ODC enzyme half-life through co-immunoprecipitation with ODC enzyme or through conventional protein purification techniques. We will thus obtain an understanding of the mechanisms by which TPA causes changes in ODC gene expression in human keratinocytes, and how, in mouse skin. TPA can cause tumor-promotion, whereas in human keratinocytes, TPA can cause cellular differentiation. This information will give us greater insight into skin cell proliferation, thereby contributing to the development of rational treatments of human epidermal disease.

佛波醇酯，12-0-十四酰基-佛波醇-13-乙酸酯（TPA）， 一种有效的肿瘤促进剂， 皮肤系统 在人类角质形成细胞中，TPA诱导 表皮分化和抑制鸟氨酸脱羧酶 (ODC)活性而不引起ODC mRNA的任何伴随变化 程度. 相反，在小鼠皮肤中，TPA是增殖剂， 并导致ODC活性、mRNA水平和基因表达水平的增加， 转录速率;这种ODC活性的增加似乎是 肿瘤促进所必需的，表明ODC的重要作用 在致癌作用中。 因此，TPA在人体内具有相反的作用， 小鼠，这些研究的目的是阐明机制 由此TPA抑制人角质形成细胞中的ODC基因表达。 随着TPA降低ODC合成速率， ODC蛋白的稳定性，我们推测TPA调控ODC基因 通过调节ODC mRNA的翻译能力和通过改变 ODC酶半衰期。 这些影响的机制将是 研究通过（1）阐明TPA对表达的影响， 在人和小鼠角质形成细胞中转染人ODC cDNA，（2） 评估ODC mRNA 51-非编码（5 '-nc）区在 通过调节TPA对ODC mRNA翻译能力的影响， 5 ′-NC区缺失体的转染，（3）阐明 TPA对ODC mRNA翻译的影响是通过蛋白质介导的 使用RNA-蛋白UV与ODC mRNA 5 '-nc区结合 交联测定，（4）确定ODC中的哪些序列 蛋白质介导其不稳定的TPA，通过转染 探针编码区缺失体和测定酶半衰期， 和（5）分离与ODC序列相互作用的蛋白质 通过以下途径介导TPA对ODC酶半衰期的影响 与ODC酶的免疫共沉淀或通过常规的 蛋白质纯化技术。 我们将获得一个 了解TPA引起ODC变化的机制 人类角质形成细胞的基因表达，以及如何在小鼠皮肤中表达。 TPA可导致肿瘤促进，而在人类角质形成细胞中，TPA 会导致细胞分化 这些信息会给我们 更深入地了解皮肤细胞增殖，从而有助于 对人类表皮的合理治疗的发展 疾病