Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy. IMAGES:

心肌细胞肥大的蛋白质加速积累特性，源于细胞蛋白质合成能力的提升（核糖体含量升高）。核糖体积累的限速步骤为核糖体RNA（rRNA）基因的转录。在去甲肾上腺素或自发性收缩诱导的新生心肌细胞肥大模型中，核糖体DNA转录因子UBF（UBF）的表达变化与核糖体生物合成速率的升高呈显著相关性。我们提出假说：UBF表达上调，是上述肥大刺激因素通过提升核糖体DNA启动子转录速率发挥作用的核心机制之一。本研究直接检测了过表达UBF对体外培养的新生心肌细胞中核糖体DNA转录的影响。对照实验中，一种新型核糖体DNA转录报告基因构建体pSMECAT，在内源rRNA基因响应肥大刺激（10^-4 M苯肾上腺素、10^-7 M内皮素及自发性收缩）时，其活性变化趋势与内源rRNA基因高度相似。当收缩停滞的心肌细胞与pSMECAT及递增剂量的UBF1表达载体共转染后，可观察到核糖体DNA转录呈剂量依赖性升高（3~5倍）。蛋白质免疫印迹（Western blot）分析证实，过表达的FLAG标签标记UBF可特异性富集于心肌细胞核内。UBF1过表达足以提升新生心肌细胞的核糖体DNA转录水平这一实验结果，为下述假说提供了直接证据：UBF的调控是心肌细胞肥大相关的核糖体生物合成增强与蛋白质积累增强的关键调控环节。IMAGES: