REGULATION OF HUMAN KERATINOCYTE GENE EXPRESSION BY TPA

佛波酯，12-0-十四酰-佛波酯(TPA)是 一种强大的肿瘤促进剂，并导致皮肤表皮增生 皮肤科系统。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 级别。相比之下，在小鼠皮肤中，TPA是一种增殖剂， 并导致ODC活性、mRNA水平和基因的增加 转录速率；这种ODC活性的增加似乎是 对肿瘤的促进是必要的，这表明ODC发挥了重要作用 在致癌方面。因此，TPA对人类和 这些研究的目的是阐明其作用机制。 从而抑制人角质形成细胞中ODC基因的表达。 由于TPA降低了ODC合成率，而且似乎也在下降 ODC蛋白的稳定性，我们认为TPA调控ODC基因 通过调节ODC mRNA的可译性和改变来表达 ODC酶半衰期。这些影响的机制将是 通过(1)阐明TPA对血管内皮细胞表达的影响 人ODC基因在人和小鼠角质形成细胞中的表达(2) 评估ODC mRNA51-非编码区(5‘-NC)在 TPA对ODC基因可译性的影响 5‘-NC区缺失体的转染，(3)阐明 TPA对ODC mRNA可译性的影响是由蛋白质介导的 利用RNA-蛋白质UV与ODC mRNA5‘-NC区结合 交联法，(4)确定ODC中的哪些序列 蛋白质通过TPA介导其失稳，通过转导 探针编码区缺失和确定酶的半衰期， 分离与ODc序列相互作用的蛋白(S) 介导TPA对ODC酶半衰期的影响 ODC酶免疫共沉淀或常规免疫沉淀 蛋白质纯化技术。因此，我们将获得一个 了解TPA引起ODC变化的机制 人类角质形成细胞中的基因表达，以及如何在小鼠皮肤中表达。 TPA可以促进肿瘤，而在人类角质形成细胞中，TPA 会导致细胞分化。这一信息将给我们 对皮肤细胞增殖有更深入的了解，从而有助于 对人类表皮合理治疗方法的发展 疾病。

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.