Jeyaseelan R, Poizat C, Baker R K, Abdishoo S, Isterabadi L B, Lyons G E, Kedes L
Institute for Genetic Medicine and the Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
J Biol Chem. 1997 Sep 5;272(36):22800-8. doi: 10.1074/jbc.272.36.22800.
Doxorubicin (Dox), a cardiotoxic antineoplastic drug, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397; Jeyaseelan, R., Poizat, C., Wu, H. Y., and Kedes, L. (1997) J. Biol. Chem. 272, 5828-5832). To determine whether this drug might interfere with the function of cardiac-specific regulatory pathways, we used a differential display strategy to clone from neonatal rat cardiomyocyte candidate mRNAs that were rapidly sensitive to Dox. We report here the identification of a constitutively expressed, cardiac-restricted, nuclear protein whose mRNA level is exquisitely sensitive to Dox. Hence we have named this protein cardiac adriamycin-responsive protein (CARP). CARP mRNA is present at the earliest stages of cardiac morphogenesis. It was detected by in situ hybridization within the cardiogenic plate of 7. 5-day post coitum (p.c.) embryos, and in 8.5-day p.c. embryos CARP transcripts are present in uniformly high levels in the myocardium. Throughout cardiac development, CARP expression is specific for the myocardium; endocardial cushions and valves exhibit only background levels of signal. Transcript levels persist but gradually decrease in neonatal, 2-week-old, and adult hearts. There were no stages when CARP mRNA could not be detected. The pattern and timing of CARP mRNA expression, including transient expression in the tongue at 14.5 days p.c., coincides with that of Nkx2.5/Csx (a putative homolog of tinman, the Drosophila melanogaster gene responsible for cardiac development). The cloned full-length 1749 nucleotide CARP cDNA encodes a 319-amino acid 40-kDa polypeptide containing five tandem ankyrin repeats. CARP appears to be the rat homolog of a previously reported human single-copy gene (C-193; Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236-10245), whose mRNA is inducible by cytokines only in human endothelial cells. CARP appears to function as a negative regulator of cardiac-specific gene expression. Overexpression of CARP in cardiomyocytes suppresses cardiac troponin C and atrial natriuretic factor transcription. Cotransfection experiments in HeLa cells indicate that CARP inhibits Nkx2.5 transactivation of atrial natriuretic factor promoter. When fused to a GAL4 DNA-binding domain, CARP has transcriptional inhibitory properties in noncardiac cells. CARP thus represents the first example of a cardiac-restricted transcriptional regulatory protein that is sensitive to Dox.
阿霉素（Dox）是一种具有心脏毒性的抗肿瘤药物，它会破坏心脏特异性的基因表达程序（仓桥，M.，杜塔，S.，杰亚西兰，R.，和凯德斯，L.（1995年）《分子与细胞生物学》15卷，6386 - 6397页；杰亚西兰，R.，普瓦扎，C.，吴，H.Y.，和凯德斯，L.（1997年）《生物化学杂志》272卷，5828 - 5832页）。为了确定这种药物是否可能干扰心脏特异性调节途径的功能，我们采用差异显示策略从新生大鼠心肌细胞中克隆对阿霉素迅速敏感的候选mRNA。我们在此报告鉴定出一种组成性表达、心脏特异性、核蛋白，其mRNA水平对阿霉素极为敏感。因此我们将这种蛋白命名为心脏阿霉素反应蛋白（CARP）。CARP mRNA在心脏形态发生的最早阶段就已存在。通过原位杂交在受精后7.5天（p.c.）胚胎的心原板中检测到它，在受精后8.5天的胚胎中，CARP转录本在心肌中以均匀的高水平存在。在整个心脏发育过程中，CARP的表达对心肌具有特异性；心内膜垫和瓣膜仅显示背景水平的信号。转录本水平在新生、2周龄和成年心脏中持续存在但逐渐降低。没有检测不到CARP mRNA的阶段。CARP mRNA表达的模式和时间，包括在受精后14.5天在舌中的短暂表达，与Nkx2.5/Csx（tinman的一个假定同源物，tinman是果蝇中负责心脏发育的基因）的模式和时间一致。克隆的全长1749个核苷酸的CARP cDNA编码一个319个氨基酸的40 kDa多肽，包含五个串联的锚蛋白重复序列。CARP似乎是先前报道的人类单拷贝基因（C - 193；朱，W.，伯恩斯，D.K.，斯韦利克，R.A.，和普雷斯凯，D.H.（1995年）《生物化学杂志》270卷，10236 - 10245页）的大鼠同源物，其mRNA仅在人内皮细胞中可被细胞因子诱导。CARP似乎作为心脏特异性基因表达的负调节因子发挥作用。在心肌细胞中过表达CARP会抑制心肌肌钙蛋白C和心房利钠因子的转录。在HeLa细胞中的共转染实验表明，CARP抑制心房利钠因子启动子的Nkx2.5反式激活。当与GAL4 DNA结合结构域融合时，CARP在非心脏细胞中具有转录抑制特性。因此，CARP代表了第一个对阿霉素敏感的心脏特异性转录调节蛋白的例子。
