Isolation and Analysis of a transcriptional repressor to cause cardiac myopathy

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是作为前体(proHB-EGF)在细胞膜上产生的，金属蛋白酶通过各种刺激来切割proHB-EGF的胞外结构域(外区脱落)。一种可溶性的HB-EGF(SHB-EGF)与EGF受体结合并激活它们。有报道称，带有proHB-EGF不可切割突变的敲入小鼠，由于出现扩张性心肌病等心脏扩张，在出生后不能长期存活。然而，ucproHB-EGF对成年小鼠的影响尚不清楚。在本报告中，ucproHB-EGF诱导的H9c2大鼠心肌成肌细胞与野生型诱导的细胞相比显著导致细胞死亡。我们还发现，细胞死亡通过增加TUNEL阳性细胞而参与了细胞的凋亡。在低氧条件下，ucproHB-EGF强烈诱导H9c2细胞死亡。我们已经证明，HB-EGF的C末端片段(HB-EGF-CTF)在脱落后移位到核膜上，并通过释放对锌指转录抑制物如髓前白血病锌指蛋白(PLZF)和B细胞淋巴瘤蛋白(Bcl06)的抑制来调节转录。我们推测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所致。然后，我们利用DNA微阵列技术筛选了与细胞死亡相关的靶基因，在UC proHB-EGF诱导的H9c2细胞中发现了几个对低氧无反应的基因。另一方面，我们使用定制的DNA微阵列平板研究了从小鼠ES细胞分化而来的心脏前体细胞中的锌指转录抑制物。结果，确定了几个用于分析的候选基因。这些数据表明，前HB-EGF分泌不足与心肌细胞缺氧性死亡有关。进一步的检查可能会揭示SET心肌病的分子机制。

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.