CREB-1α is recruited to and mediates upregulation of the cytochrome c promoter during enhanced mitochondrial biogenesis accompanying skeletal muscle differentiation
CREB-1α is recruited to and mediates upregulation of the cytochrome c promoter during enhanced mitochondrial biogenesis accompanying skeletal muscle differentiation
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DOI：
10.1128/mcb.00980-07
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发表时间：
2008-04-01
影响因子：
5.3
通讯作者：
Goffart, Steffi
中科院分区：
文献类型：
--
作者：
Franko, Andras;Mayer, Sabine;Goffart, Steffi
为了进一步了解协调编码线粒体蛋白的核基因表达的途径，我们研究了成肌细胞分化为肌管期间的线粒体生物发生。这一耗能过程伴随着 ATP 周转率增加五倍，而线粒体活性增加八倍。虽然未观察到线粒体 DNA 拷贝数发生变化，但 mRNA；以及细胞核编码的细胞色素 c、细胞色素 c 氧化酶亚基 IV 和线粒体转录因子 A (TFAM) 的蛋白质以及总细胞 RNA 和蛋白质水平增加。通过荧光素酶报告基因、结合亲和力、电泳迁移率变动分析以及诱变研究对细胞色素 c 启动子进行详细分析，揭示了环 AMP 响应元件结合蛋白 1 (CREB-1) 对于启动子激活的关键作用。通过使用特异性抗体和定量逆转录 PCR 观察两种 CREB-1 亚型的表达，结果显示从成肌细胞中磷酸化的 CREB-1 Delta 转变为肌管中磷酸化的 CREB-1 α 蛋白，而 mRNA 比例保持不变。染色质免疫沉淀测定证实 CREB-lot 优先与肌管中的细胞色素 c 启动子原位结合。组成型活性和显性失活形式的过度表达支持了 CREB-1 在调节肌发生过程中编码线粒体蛋白的基因表达中的关键作用，也可能在增强线粒体生物发生的其他情况下发挥关键作用。
To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs; as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1 Delta in myoblasts to phosphorylated CREB-1 alpha protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-lot in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.