Zheng Zhenlin, Wang Zhong-Min, Delbono Osvaldo
Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
J Physiol. 2002 Apr 15;540(Pt 2):397-409. doi: 10.1113/jphysiol.2001.013464.
Several factors, such as Ca(2+), trophic factors and ageing, regulate dihydropyridine-sensitive receptor (DHPR) alpha(1) subunit expression. However, basic mechanisms of DHPR alpha(1S) expression are unknown. To better understand the regulatory elements that control transcription, the 1.2 kb 5'-flanking region fragment immediately upstream of the mouse L-type Ca(2+) channel or DHPR alpha(1S) gene was isolated and sequenced. Luciferase reporter constructs driven by different promoter regions of mouse DHPR alpha(1S) gene were used for transient transfection assays in muscle C2C12 cells. In these preparations we found that three regions corresponding to CREB, GATA-2 and SOX-5 consensus sequence within the 5'-flanking region of the DHPR alpha(1S) gene are important for DHPR alpha(1S) gene transcription. Antisense oligonucleotides against CREB, GATA-2 and SOX-5 significantly reduced charge movement in C2C12 cells. Charge movement was recorded in the whole-cell configuration of the patch clamp technique. Results from cells transfected with antisense (AS) and sense (S) oligonucleotides and nontransfected cells were compared. Charge movement experiments were fitted to a Boltzmann equation. Maximum charge movement (Q(max)) (nC microF(-1), mean +/- S.E.M.) for S- and AS-CREB was 70.3 +/- 2.9 and 52.8 +/- 3.3, respectively (P < 0.05). The same parameter for S- and AS-GATA-2 was 71.3 +/- 3.9 and 48.2 +/- 2.3, respectively (P < 0.05) and for S- and AS-SOX-5 was 70.4 +/- 4.2 and 45.1 +/- 3.2, respectively (P < 0.05). Values recorded in cells transfected with sense S-CREB, S-GATA-2 and S-SOX-5 oligonucleotides were not significantly different from those recorded in nontransfected cells. This study demonstrates that the transcription factors CREB, GATA-2 and SOX-5 play a significant role in the expression of the skeletal muscle DHPR or L-type Ca(2+) channel alpha(1S).
多种因素，如钙离子、营养因子和衰老，可调节二氢吡啶敏感受体（DHPR）α1亚基的表达。然而，DHPRα1S表达的基本机制尚不清楚。为了更好地理解控制转录的调控元件，我们分离并测序了小鼠L型钙离子通道或DHPRα1S基因上游紧邻的1.2kb 5'侧翼区域片段。由小鼠DHPRα1S基因不同启动子区域驱动的荧光素酶报告基因构建体用于肌肉C2C12细胞的瞬时转染实验。在这些实验中，我们发现DHPRα1S基因5'侧翼区域内与CREB、GATA-2和SOX-5共有序列相对应的三个区域对DHPRα1S基因转录很重要。针对CREB、GATA-2和SOX-5的反义寡核苷酸显著降低了C2C12细胞中的电荷移动。电荷移动采用膜片钳技术的全细胞模式进行记录。比较了转染反义（AS）和正义（S）寡核苷酸的细胞以及未转染细胞的结果。电荷移动实验数据用玻尔兹曼方程拟合。S-CREB和AS-CREB的最大电荷移动（Q(max)）（nC μF-1，平均值±标准误）分别为70.3±2.9和52.8±3.3（P<0.05）。S-GATA-2和AS-GATA-2的相同参数分别为71.3±3.9和48.2±2.3（P<0.05），S-SOX-5和AS-SOX-5的相同参数分别为70.4±4.2和45.1±3.2（P<0.05）。转染正义S-CREB、S-GATA-2和S-SOX-5寡核苷酸的细胞记录值与未转染细胞的记录值无显著差异。本研究表明，转录因子CREB、GATA-2和SOX-5在骨骼肌DHPR或L型钙离子通道α1S的表达中起重要作用。
