VITAMIN A INFLUENCE ON INTERFERON-GAMMA GENE EXPRESSION

维生素 A 对干扰素-γ 基因表达的影响

• 批准号: 2146076
• 负责人: COLLEEN Elizabeth HAYES
• 金额: $ 11.73万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-20 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2146076
• 关键词: T lymphocyte; antibody; cell growth regulation; clone cells; enzyme linked immunosorbent assay; gene induction /repression; genetic regulation; genetic transcription; genetic translation; hamsters; immunochemistry; interferon gamma; interferon inducers; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; northern blottings; nucleic acid probes; polymerase chain reaction; protein isoforms; retinoid binding proteins; retinoids

Surprisingly, vitamin A-deficient mice undergoing an infection, or immunized with an antigen, had T lymphocytes that secreted IFNgamma at a 10-fold higher rate than controls. The T cells from A-deficient mice functioned poorly as stimulators of B cell antibody responses; there was a low helper T cell frequency and the cells underproduced the lymphokines IL-4 and IL-5. Repletion with retinoic acid, a vitamin A metabolite, sharply downregulated IFNgamma, restored the helper T cell frequency, and returned antibody responses to normal levels in vitro. Retinoid repletion also restored antibody responses in vivo. Cloned mouse MD13-10 T cells mimic freshly explanted immune T cells with respect to IFNgamma production. When MD 13-10 T cells are retinoid depleted and stimulated, they overproduce IFNgamma, and retinoic acid directly downregulates the IFNgamma secretion rate. We found an unexpected retinoic acid receptor- alpha (RARalpha) transcript and a novel RARalpha-immunoreactive protein in MD13-10 T cells and other T cells. Together, our findings lead us to propose as a working hypothesis that retinoic acid, acting through an undefined mechanism possibly involving this novel RARalpha isoform, specifically downregulates T cell IFNgamma production. The experiments proposed here will test our hypothesis by studying the biochemistry of retinoid-mediated IFNgamma regulation in cloned MD 13- 10 T cells. We propose to define the retinoid-sensitive IFNgamma induction pathway, to explore whether retinoid-mediated control is at the IFNgamma transcript or protein level, and to analyze the dependence of regulation on macromolecular synthesis. We propose to characterize the T cell RARalpha transcripts by Northern blotting with exon-specific probes, and sequencing of novel 5' and/or 3' cDNA ends. We will investigate the putative RARalpha proteins using immunochemistry. We plan to express and purify the novel RARalpha N-terminal fragments, and raise antibodies. We will use the antibodies to develop RARalpha isoform-specific ELISA, retinoid-binding, and DNA-binding assays and apply them to MD13-10 T cell proteins. The proposed research is significant in that it will provide new information about a regulatory relationship between two potent controllers of immune function and cell growth. IFNgamma regulates immunity, autoimmunity, hemopoiesis, viral replication, and malignant cell growth. Retinoic acid also regulates immunity, and normal and malignant cell growth and differentiation. The powerful anticancer and immunoregulatory activities of both molecules emphasize the importance of investigating how retinoic acid downregulates IFNgamma gene expression.

令人惊讶的是，维生素A缺乏的小鼠经历感染，或 用抗原免疫，T淋巴细胞分泌IFN γ， 10-比对照组高出一倍。来自A缺陷小鼠的T细胞 作为B细胞抗体应答的刺激物功能不佳; 辅助性T细胞的频率较低，细胞产生的淋巴因子不足， IL-4和IL-5。补充维生素A代谢物维甲酸， 显著下调IFN γ，恢复辅助性T细胞频率， 在体外将抗体应答恢复到正常水平。类维生素A充盈 也恢复了体内的抗体反应。克隆小鼠MD 13 -10 T细胞 就IFN γ而言，模拟新鲜接种的免疫T细胞 生产当MD 13-10 T细胞被类维生素A耗尽并被刺激时， 它们过度产生IFN γ，视黄酸直接下调 IFN γ分泌速率。我们发现了一种意想不到的视黄酸受体- 一种新的RAR α免疫反应性蛋白， MD 13 -10 T细胞和其它T细胞。综合起来，我们的发现将我们引向 提出一个工作假设，视黄酸，通过一个 可能涉及这种新的RAR α亚型的未定义机制， 特异性下调T细胞IFN γ的产生。实验 这里提出的将通过研究生物化学来验证我们的假设。 在克隆的MD 13- 10 T细胞中类维生素A介导的IFN γ调节。我们 建议定义类维生素A敏感的IFN γ诱导途径， 探索类维生素A介导的控制是否在IFN γ转录物或 蛋白质水平，并分析调节的依赖性， 大分子合成我们建议将T细胞RAR α 用外显子特异性探针通过北方印迹和测序检测转录物 新的5'和/或3' cDNA末端。我们将研究假定的RAR α 蛋白质免疫化学。我们计划表达和净化小说 RAR α N-末端片段，并产生抗体。我们将使用 抗体以开发RAR α亚型特异性ELISA，类维生素A结合， 和DNA结合测定，并将它们应用于MD 13 -10 T细胞蛋白。的 这项研究的意义在于它将提供新的信息， 关于两个免疫系统控制者之间的调节关系 功能和细胞生长。IFN γ调节免疫，自身免疫， 造血、病毒复制和恶性细胞生长。维甲酸 还调节免疫力，以及正常和恶性细胞生长， 分化强大的抗癌和免疫调节活性 强调了研究维甲酸是如何 酸下调IFN γ基因表达。