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Isolation and Analysis of a transcriptional repressor to cause cardiac myopathy

引起心肌病的转录抑制因子的分离和分析

基本信息

批准号：
18590298
负责人：
INOUE Hirofumi
金额：
$ 2.65万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590298/
关键词：
cardiomyocyte growth factor cardiac myocytosis cell death transcriptional repressor HB-EGF 分化誘導転写抑制因子

项目摘要

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a type of transmembrane protein. HB-EGF is produced as a precursor (proHB-EGF) in cell membrane and metalloproteases cleave the ectodomain of proHB-EGF (ectodomain shedding) by various stimulations. A soluble form of HB-EGF (sHB-EGF) binds EGF receptors and activates them. It has been reported that a knock-in mouse with uncleavable mutant of proHB-EGF can not survive for a long term after birth because of onset of dilatation of a heart such as dilatation cardiomyopathy. However, Effects of ucproHB-EGF on adult mice remain unclear. In this report, ucproHB-EGF-induced H9c2 rat cardiomyoblasts significantly lead to cell death as compared with wild type-induced cells. And we also found that the cell death involved apoptosis by increasing TUNEL positive cells. Under hypoxia, ucproHB-EGF strongly induced cell death of H9c2. We have already shown that C-terminal fragment of proHB-EGF (HB-EGF-CTF) after shedding translocates to nuclear membrane and regulate transcription by releasing suppression of zinc finger transcriptional repressors such as premyeloid leukemia zinc finger protein (PLZF) and B-cell lymphoma protein (Bcl06). We hypothesized that uc proHB-EGF-induced cell death was due to failure of transcriptional response by insufficiency of HB-EGF-CTF production. Then, we performed DNA microarray assay to identify cell death-associated target genes, and found several genes not to response to hypoxia in uc proHB-EGF-induced H9c2 cells. In other hand, we investigated zinc finger transcriptional repressors in cardiac precursor cells differentiated from mouse ES cells with custom DNA microarray plates. As a result, several candidate genes for analysis were identified. These data suggest that insufficient shedding of proHB-EGF is associated with hypoxic cell death in cardiomyocytes. Further examinations could unveil a molecular mechanism of cardiomyopathy on set.

肝素结合表皮生长因子样生长因子（HB-EGF）是一种跨膜蛋白。 HB-EGF 在细胞膜中作为前体 (proHB-EGF) 产生，金属蛋白酶通过各种刺激裂解 proHB-EGF 的胞外域（胞外域脱落）。可溶形式的 HB-EGF (sHB-EGF) 结合 EGF 受体并激活它们。据报道，带有proHB-EGF不可切割突变体的敲入小鼠在出生后不能长期存活，因为出现心脏扩张，例如扩张性心肌病。然而，ucproHB-EGF 对成年小鼠的影响仍不清楚。在本报告中，与野生型诱导的细胞相比，ucproHB-EGF 诱导的 H9c2 大鼠心肌细胞显着导致细胞死亡。我们还发现细胞死亡是通过增加TUNEL阳性细胞而导致细胞凋亡。缺氧条件下，ucproHB-EGF强烈诱导H9c2细胞死亡。我们已经证明，proHB-EGF (HB-EGF-CTF) 的 C 端片段在脱落后易位到核膜，并通过释放对锌指转录抑制因子（如早髓性白血病锌指蛋白 (PLZF) 和 B 细胞淋巴瘤蛋白 (Bcl06)）的抑制来调节转录。我们假设 uc proHB-EGF 诱导的细胞死亡是由于 HB-EGF-CTF 产生不足导致转录反应失败。然后，我们进行了DNA微阵列分析来鉴定与细胞死亡相关的靶基因，并发现了uc proHB-EGF诱导的H9c2细胞中的几个对缺氧没有反应的基因。另一方面，我们使用定制的 DNA 微阵列板研究了从小鼠 ES 细胞分化而来的心脏前体细胞中的锌指转录抑制因子。结果，鉴定了几个用于分析的候选基因。这些数据表明 proHB-EGF 脱落不足与心肌细胞缺氧细胞死亡有关。进一步的检查可以揭示心肌病的分子机制。