ALPHA-1 ADRENERGIC REGULATION OF CARDIAC GENE EXPRESSION

描述(改编自申请人的摘要)：一个重要的 心肌生物学中的问题是细胞外信号是如何激活的 心脏基因转录。一种基因激活方法可能提供一种 在活体中有目的地改变心脏表型的分子开关。 申请人正在研究激活心肌细胞生长和 儿茶酚胺通过A1-肾上腺素能作用于基因转录 受体(AR)。重点一直放在两种收缩的调节上 蛋白质同基因、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”的范例，在许多情况下是保守的 体内和培养的肥大模型。通过研究bMHC和 在培养的心肌细胞中的SkACT启动子，申请人已经 鉴定出一个DNA序列，即增强子核心/M-CAT元件，即 通过A1-AR刺激激活这些启动子的反应元件。 申请人还确定了与之相互作用的转录因子。 有了这个反应元件，转录增强因子-1(TEF-1)。 虽然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对肿瘤细胞的生长是不可或缺的 活体内的胎儿心脏。申请人已从大鼠心脏克隆了TEF-1 肌细胞。他发现了7种TEF-1亚型，其中只有几种是 以前被识别的，可能是通过替代剪接产生的。通过 核糖核酸酶保护实验，一种异构体的mRNA在成人中表达 心肌和骨骼肌，但不在脑或肝脏。当共同- 转染b-MHC启动子后，五种TEF-1亚型被激活， 和两个抑制，这两个作用都是M-cat位点依赖的。因此， 心肌细胞是第一种细胞类型，通过 已发现TEF-1，心脏可能含有肌肉特异性TEF-1 异构体。A1-AR激动剂对心肌细胞的刺激作用 B-蛋白激酶C(PCK)结合到细胞核，导致TEF-1的磷酸化 增加TEF-1的DNA结合量和蛋白质含量。B-PCK刺激 M-CAT元件，并在体外磷酸化TEF-1。因此，这些 结果表明，在该模型中，A1-AR刺激心肌细胞 表面激活b-PKC并使TEF-1磷酸化。这将激活TEF-1 并增加其DNA结合和/或反式激活功能。 激活的TEF-1然后增加其目标基因的转录。二 这项提案将探讨假设。首先，TEF-1是 通过PKC被A1-AR刺激磷酸化，这激活了它的 功能。其次，TEF-1的磷酸化机制可用于 改变心脏分子表型，不依赖于受体，在 培养的心肌细胞，以及完整的心脏。为了测试这些想法，有两个 问题将会被问到。首先，是否存在心脏特异的TEF-1 异构体，这些在DNA结合、反式激活和 翻译？第二，b-PKC的刺激是否会产生磷酸化？ TEF-1上的特定残基？这些残留物是相同的吗？ 被A1-AR磷酸化？PCK在这方面是独一无二的吗？最后，做 心肌细胞培养中TEF-1活性变化及转录变化 在完好无损的动物身上？

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?