ALPHA-1 ADRENERGIC REGULATION OF CARDIAC GENE EXPRESSION

心脏基因表达的 ALPHA-1 肾上腺素调节

• 批准号: 2220319
• 负责人: PAUL C SIMPSON
• 金额: $ 27.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2220319
• 关键词: RNase protection assay; actins; alpha adrenergic receptor; catecholamines; gene expression; genetic enhancer element; genetic promoter element; genetic transcription; heart cell; laboratory rat; messenger RNA; myosins; protein isoforms; protein kinase C; tissue /cell culture; transfection; ventricular hypertrophy

DESCRIPTION (adapted from the applicant's abstract): An important problem in myocardial biology is how extracellular signals activate cardiac gene transcription. A gene activation method might provide a molecular switch to purposefully alter the cardiac phenotype in vivo. The applicant is studying the activation of cardiac myocyte growth and gene transcription by catecholamines acting through an a1-adrenergic receptor (AR). The focus has been on regulation of two contractile protein isogenes, the b-myosin heavy chain (MHC) and skeletal a-actin (skACT), since they exemplify a "fetal program", conserved in many models of hypertrophy in vivo and in culture. By studying the bMHC and skACT promoters in cultured cardiac myocytes, the applicant has identified a DNA sequence, enhancer core/M-CAT element, that is a response element for activation of these promoters by a1-AR stimulation. The applicant also has identified the transcription factor that interacts with this response element, transcriptional enhancer factor-1 (TEF-1). Although TEF-1 was cloned originally in the context of a viral promoter, gene knockout has shown that TEF-1 is indispensable for growth of the fetal heart in vivo. The applicant has cloned TEF-1 from rat cardiac myocytes. He finds seven TEF-1 isoforms, only a few of which had been recognized previously, probably produced by alternative splicing. By RNase protection assay, the mRNA for one isoform is expressed in adult cardiac and skeletal muscle, but not in brain or liver. When co- transfected with a b-MHC promoter, five of the TEF-1 isoforms activate, and two repress, and both actions are M-CAT site-dependent. Thus, cardiac myocytes are the first cell type in which transactivation by TEF-1 has been seen and heart may contain a muscle-specific TEF-1 isoform. Stimulation of myocytes with an a1-AR agonists translocates b-protein kinase C (PCK) to the nucleus, causes phosphorylation of TEF-1 and increases TEF-1 DNA binding and protein amount. b-PCK stimulates the M-CAT element and phosphorylates TEF-1 in vitro. Thus, these results suggest a model wherein stimulation of a a1-AR at the myocytes surface activates b-PKC and phosphorylates TEF-1. This activates TEF-1 and increases its DNA binding and/or transactivating functions. Activated TEF-1 then increases transcription of its target genes. Two hypotheses will be explored in this proposal. First, TEF-1 is phosphorylated by a1-AR stimulation through PKC and this activates its functions. Secondly, The TEF-1 phosphorylation mechanism can be used to alter the cardiac molecular phenotype independent of the receptor, in cultured myocytes, and in intact heart. To test these ideas, two questions will be asked. First, are there cardiac-specific TEF-1 isoforms, and do these differ in DNA binding, transactivation, and translation? Secondly, does b-PKC stimulation produce phosphorylation of specific residues on TEF-1? Are these the same residues phosphorylated by a1-AR? Is PCK unique in this respect? Finally, do alteration of TEF-1 activity change transcription in myocyte culture and in the intact animal?

描述（改编自申请人的摘要）：重要的 心肌生物学的一个问题是细胞外信号如何激活 心脏基因转录基因激活方法可以提供 分子开关来有目的地改变体内心脏表型。 申请人正在研究心肌细胞生长的激活， 通过α 1-肾上腺素能受体作用的儿茶酚胺的基因转录 受体（AR）。重点是调节两种收缩性 蛋白质同源基因，b-肌球蛋白重链（MHC）和骨骼α-肌动蛋白 （skACT），因为它们是一个“胎儿程序”，在许多 在体内和培养中的肥大模型。通过研究bMHC和 skACT启动子，申请人已经 确定了一个DNA序列，增强子核心/M-CAT元件，这是一个 通过a1-AR刺激激活这些启动子的反应元件。 申请人还鉴定了与转录因子相互作用的转录因子。 转录增强因子-1（TEF-1）。 尽管TEF-1最初是在病毒启动子的背景下克隆的， 基因敲除表明，TEF-1是不可或缺的生长， 体内胎心。申请人已从大鼠心脏中克隆了TEF-1 肌细胞他发现了七种TEF-1亚型，其中只有少数被发现。 以前认识的，可能是由选择性剪接产生的。通过 RNA酶保护测定，一种亚型的mRNA在成体中表达。 心脏和骨骼肌，但不在大脑或肝脏中。当共同- 用b-MHC启动子转染后，TEF-1的五种同种型被激活， 和两个抑制，并且这两个作用都是M-CAT位点依赖的。因此，在本发明中， 心肌细胞是第一种通过反式激活的细胞类型 TEF-1已被发现，心脏可能含有肌肉特异性TEF-1 同种型用α 1-AR激动剂刺激肌细胞易位 b-蛋白激酶C（PCK）进入细胞核，导致TEF-1磷酸化 并增加TEF-1 DNA结合和蛋白量。b-PCK刺激 M-CAT元件并在体外磷酸化TEF-1。因此这些 结果提示了一种模型，其中在肌细胞处刺激α 1-AR 表面活化b-PKC并磷酸化TEF-1。这激活了TEF-1 并增加其DNA结合和/或反式激活功能。 激活的TEF-1然后增加其靶基因的转录。两 本提案将探讨各种假设。首先，TEF-1是 通过PKC被α 1-AR刺激磷酸化，这激活了它的 功能协调发展的其次，TEF-1磷酸化机制可用于 不依赖于受体改变心脏分子表型， 培养的心肌细胞和完整的心脏。为了验证这些想法，两个 会被问到问题首先，是否存在心脏特异性TEF-1 异构体，这些在DNA结合，反式激活和 翻译？第二，b-PKC刺激是否产生磷酸化 TEF-1上的特定残留物这些残留物 被α 1-AR磷酸化？PCK在这方面是独一无二的吗？最后，做 TEF-1活性的改变改变了心肌细胞培养中的转录， 在完整的动物？