Katoh Y, Loukianov E, Kopras E, Zilberman A, Periasamy M
Molecular Cardiology Laboratory, University of Cincinnati College of Medicine, Ohio 45267-0542.
J Biol Chem. 1994 Dec 2;269(48):30538-45.
Despite the importance of smooth muscle cell proliferation in vascular pathophysiological states, the mechanisms regulating smooth muscle cell growth and differentiation are poorly understood. Previous studies have shown that adult rabbit smooth muscles express two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are generated through alternative RNA splicing from a single smooth muscle MHC (SMHC) gene. In the present study, we isolated and characterized the rabbit SMHC gene promoter. DNA sequence analysis of the upstream region of the SMHC gene revealed several putative cis-DNA regulatory elements proximal to the transcription start site. Most notably, cis-acting regulatory elements that closely resemble CC(A/T)6GG (CArG box) and myocyte enhancer binding factor 2 (MEF-2)-type sequence motifs were found in the SMHC 5'-flanking region. In addition, six E-box motifs were found in the 5'-flanking region of the SMHC gene between -374 and -2109 base pairs from the transcription start site. A series of transient transfection assays using SMHC promoter deletion constructs indicated that a promoter fragment extending to 2266 base pairs upstream of the transcription start site has the highest reporter activity in cultured rat aortic smooth muscle cells. Gel mobility shift analyses using the MEF-2-like sequence located at -1540 revealed a specific DNA protein complex, whereas the CArG-like element located at -1275 did not show protein binding. The SMHC promoter construct, p509-CAT, which included neither the CArG- nor MEF-2-type motifs, conferred 32% of chloramphenicol acetyltransferase activity in the same cells, whereas the construct p188-CAT, which contained the minimal promoter elements (TATA box), was significantly less active (7%; 2.0-fold over background). This is the first report describing the promoter elements of a gene whose expression is restricted to smooth muscle cells.
尽管平滑肌细胞增殖在血管病理生理状态中具有重要意义，但调节平滑肌细胞生长和分化的机制仍知之甚少。先前的研究表明，成年兔平滑肌表达两种类型的肌球蛋白重链（MHC）同工型，即SM1和SM2，它们是通过从单个平滑肌MHC（SMHC）基因进行可变RNA剪接产生的。在本研究中，我们分离并鉴定了兔SMHC基因启动子。对SMHC基因上游区域的DNA序列分析揭示了转录起始位点附近的几个假定的顺式DNA调控元件。最值得注意的是，在SMHC 5'侧翼区域发现了与CC（A/T）6GG（CArG框）和心肌细胞增强子结合因子2（MEF-2）型序列基序非常相似的顺式作用调控元件。此外，在SMHC基因5'侧翼区域，从转录起始位点起-374至-2109碱基对之间发现了六个E-box基序。使用SMHC启动子缺失构建体进行的一系列瞬时转染试验表明，延伸至转录起始位点上游2266碱基对的启动子片段在培养的大鼠主动脉平滑肌细胞中具有最高的报告基因活性。使用位于-1540的MEF-2样序列进行的凝胶迁移率变动分析揭示了一种特异性DNA-蛋白质复合物，而位于-1275的CArG样元件未显示蛋白质结合。既不包含CArG也不包含MEF-2型基序的SMHC启动子构建体p509-CAT在相同细胞中赋予了32%的氯霉素乙酰转移酶活性，而包含最小启动子元件（TATA框）的构建体p188-CAT活性明显较低（7%；比背景高2.0倍）。这是第一份描述其表达仅限于平滑肌细胞的基因的启动子元件的报告。
