Chen Y H, Layne M D, Watanabe M, Yet S F, Perrella M A
Pulmonary and Critical Care and Cardiovascular Divisions, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.
J Biol Chem. 2001 Dec 14;276(50):47658-63. doi: 10.1074/jbc.M108678200. Epub 2001 Oct 17.
The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. Aortic preferentially expressed gene-1 (APEG-1), a VSMC-specific gene, is expressed highly in differentiated but not in dedifferentiated VSMC. Previously, we identified an E-box element in the mouse APEG-1 proximal promoter, which is essential for VSMC reporter activity. In this study, we investigated the role of upstream stimulatory factors (USF) in the regulation of APEG-1 transcription via this E-box element. By electrophoretic mobility shift assays, recombinant USF1 and USF2 homo- and heterodimers bound specifically to the APEG-1 E-box. Nuclear extracts prepared from primary cultures of rat aortic smooth muscle cells exhibited specific USF1 and USF2 binding to the APEG-1 E-box. To investigate the binding properties of USF during VSMC differentiation, nuclear extracts were prepared from the neural crest cell line, MONC-1, which differentiates into VSMC in culture. Maximal USF1 and USF2 protein levels and binding to the APEG-1 E-box occurred 3 h after the differentiation of MONC-1 cells was initiated. Co-transfection experiments demonstrated that dominant negative USF repressed APEG-1 promoter activity, and USF1, but not USF2, transactivated the APEG-1 promoter. Our studies demonstrate that USF factors contribute to the regulation of APEG-1 expression and may influence the differentiation of VSMC.
血管平滑肌细胞（VSMC）的表型调节在动脉粥样硬化的发病机制中起着核心作用。主动脉优先表达基因-1（APEG-1）是一种VSMC特异性基因，在分化的VSMC中高表达，而在去分化的VSMC中不表达。此前，我们在小鼠APEG-1近端启动子中鉴定出一个E盒元件，它对VSMC报告基因活性至关重要。在本研究中，我们研究了上游刺激因子（USF）通过该E盒元件对APEG-1转录的调节作用。通过电泳迁移率变动分析，重组USF1和USF2同二聚体及异二聚体特异性结合到APEG-1 E盒上。从大鼠主动脉平滑肌细胞原代培养物制备的核提取物显示出USF1和USF2与APEG-1 E盒的特异性结合。为了研究VSMC分化过程中USF的结合特性，从神经嵴细胞系MONC-1制备核提取物，该细胞系在培养中可分化为VSMC。在MONC-1细胞开始分化后3小时出现最大的USF1和USF2蛋白水平以及与APEG-1 E盒的结合。共转染实验表明，显性负性USF抑制APEG-1启动子活性，而USF1而非USF2可激活APEG-1启动子。我们的研究表明，USF因子有助于调节APEG-1的表达，并可能影响VSMC的分化。
