Cohen-Barak Orit, Yi Zanhua, Hagiwara Nobuko, Monzen Koshiro, Komuro Issei, Brilliant Murray H
Department of Pediatrics, The University of Arizona College of Medicine, Steele Memorial Children's Research Center 1501 N. Campbell Avenue, Tucson, AZ 85724, USA.
Nucleic Acids Res. 2003 Oct 15;31(20):5941-8. doi: 10.1093/nar/gkg807.
A mouse mutation (p100H/p100H) has been identified that is associated with cardioskeletal myopathy, heart block, delayed growth and early postnatal death. The gene that is disrupted in this mutation encodes the transcription factor Sox6. P19CL6 cells were used as an in vitro cardiomyocyte differentiation system and revealed that Sox6 is expressed exclusively when the cells are committed to differentiate to beating cardiac myocytes. We used the yeast two-hybrid system to identify the Prtb (Proline-rich transcript of the brain) protein as a Sox6 interactor, and subsequently confirmed the interaction by co-immunoprecipitation. Prtb expression in P19CL6 cells increased with differentiation to beating cardiomyocytes. Using the P19CL6 cells stably transfected with noggin, an antagonist of BMP (Bone Morphogenic Protein), we found that BMP expression is required for Sox6 expression in cardiomyocyte differentiation. Surprisingly, the expression of the alpha1c-subunit gene of the L-type Ca2+ channel decreased in P19CL6 cells as they differentiated to beating cardiac cells. Ectopic expression of Sox6 or Prtb alone in P19CL6 cells caused down-regulation of L-type Ca2+ alpha1c expression, but when Sox6 and Prtb were co-transfected to the cells, L-type Ca2+ alpha1c remained at basal levels. A similar relationship of Sox6 and L-type Ca2+ alpha1c expression was seen in vivo (comparing wild-type and p(100H)/p(100H) mutant mice). Thus, Sox6 is within the BMP pathway in cardiac differentiation, interacts with Prtb and may play a critical role in the regulation of a cardiac L-type Ca2+ channel.
已鉴定出一种小鼠突变体（p100H/p100H），其与心肌骨骼肌病、心脏传导阻滞、生长迟缓和出生后早期死亡相关。在此突变中被破坏的基因编码转录因子Sox6。P19CL6细胞被用作体外心肌细胞分化系统，结果显示，只有当细胞开始分化为跳动的心肌细胞时，Sox6才会表达。我们使用酵母双杂交系统鉴定出富含脯氨酸的脑转录本（Prtb）蛋白为Sox6相互作用蛋白，随后通过免疫共沉淀证实了这种相互作用。P19CL6细胞中Prtb的表达随着向跳动心肌细胞的分化而增加。使用稳定转染了骨形态发生蛋白（BMP）拮抗剂头蛋白（noggin）的P19CL6细胞，我们发现BMP表达是心肌细胞分化过程中Sox6表达所必需的。令人惊讶的是，P19CL6细胞在分化为跳动的心脏细胞时，L型Ca2+通道的α1c亚基基因表达下降。单独在P19CL6细胞中异位表达Sox6或Prtb会导致L型Ca2+α1c表达下调，但当将Sox6和Prtb共转染到细胞中时，L型Ca2+α1c保持在基础水平。在体内（比较野生型和p(100H)/p(100H)突变小鼠）也观察到了Sox6与L型Ca2+α1c表达之间的类似关系。因此，Sox6处于心脏分化的BMP信号通路中，与Prtb相互作用，并可能在心脏L型Ca2+通道的调节中起关键作用。
