Monzen Koshiro, Zhu Weidong, Kasai Hiroki, Hiroi Yukio, Hosoda Toru, Akazawa Hiroshi, Zou Yunzeng, Hayashi Doubun, Yamazaki Tsutomu, Nagai Ryozo, Komuro Issei
Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan.
Biochem Biophys Res Commun. 2002 Nov 8;298(4):493-500. doi: 10.1016/s0006-291x(02)02497-x.
A homeobox-containing transcription factor Csx/Nkx2-5 is an important regulator of cardiac development. Many different human CSX/NKX2-5 mutations have been reported to cause congenital heart disease. We here examined the effects of three representative CSX/NKX2-5 mutations on cardiomyocyte differentiation and death with the use of the P19CL6 cardiomyogenic cell lines. Stable overexpression of wild-type CSX/NKX2-5 enhanced expression of cardiac-specific genes such as MEF2C and MLC2v, the promoter activity of the atrial natriuretic peptide gene, and the terminal differentiation of P19CL6 into cardiomyocytes, while all CSX/NKX2-5 mutants attenuated them by different degrees. When exposed to H(2)O(2) or cultured without change of the medium, many differentiated P19CL6 cells overexpressing the mutants, especially the mutant which lacks the carboxyl terminal region just after the homeodomain, were dead, while most of the cells overexpressing wild-type CSX/NKX2-5 survived. Overexpression of the carboxyl terminus-deleted mutant down-regulated expression of an anti-apoptotic protein Bcl-x(L) and up-regulated that of a pro-apoptotic protein CAS, while in the cells overexpressing wild-type CSX/NKX2-5, expression of a pro-apoptotic protein RIP was reduced. Furthermore, overexpression of wild-type CSX/NKX2-5 decreased the number of H(2)O(2)-induced TUNEL-positive cultured cardiomyocytes of neonatal rats, whereas overexpression of the mutants enhanced it. These results suggest that Csx/Nkx2-5 not only regulates expression of cardiac-specific genes but protects cardiomyocytes from stresses and that cell death may be another cause for the cardiac defects induced by human CSX/NKX2-5 mutations.
含同源异型框的转录因子Csx/Nkx2-5是心脏发育的重要调节因子。据报道，许多不同的人类CSX/NKX2-5突变会导致先天性心脏病。我们在此利用P19CL6心肌生成细胞系研究了三种代表性CSX/NKX2-5突变对心肌细胞分化和死亡的影响。野生型CSX/NKX2-5的稳定过表达增强了心肌特异性基因（如MEF2C和MLC2v）的表达、心钠素基因的启动子活性以及P19CL6向心肌细胞的终末分化，而所有CSX/NKX2-5突变体均不同程度地减弱了这些作用。当暴露于H₂O₂或在培养基不变的情况下培养时，许多过表达突变体的分化P19CL6细胞，尤其是缺乏同源异型域后羧基末端区域的突变体，会死亡，而过表达野生型CSX/NKX2-5的大多数细胞存活。羧基末端缺失突变体的过表达下调了抗凋亡蛋白Bcl-x(L)的表达，上调了促凋亡蛋白CAS的表达，而在过表达野生型CSX/NKX2-5的细胞中，促凋亡蛋白RIP的表达降低。此外，野生型CSX/NKX2-5的过表达减少了H₂O₂诱导的新生大鼠培养心肌细胞中TUNEL阳性细胞的数量，而突变体的过表达则增加了该数量。这些结果表明，Csx/Nkx2-5不仅调节心肌特异性基因的表达，还能保护心肌细胞免受应激，细胞死亡可能是人类CSX/NKX2-5突变诱导心脏缺陷的另一个原因。
