Simonson M S, Walsh K, Kumar C C, Bushel P, Herman W H
Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Am J Physiol. 1995 Apr;268(4 Pt 2):F760-9. doi: 10.1152/ajprenal.1995.268.4.F760.
Mesangial cells express smooth muscle alpha-actin (SM alpha-actin) in response to glomerular injury in vivo, and SM alpha-actin gene expression serves as a genetic marker characterizing the activated phenotype of mesangial cells. We used a molecular genetic approach to analyze the SM alpha-actin promoter and evaluate transcriptional mechanisms that might direct the genetic switch of mesangial cells to the activated phenotype. The sequence spanning -894 to +1 of the SM alpha-actin promoter directed high levels of transcription that were attenuated in serum-restricted cells and upregulated upon treatment with serum or endothelin-1. Deletional analysis revealed a core promoter fragment, from positions -122 to +1, that was necessary and sufficient for transcription. This core activity was modulated by upstream sequences between -670 and -122. The 122-bp core promoter contains two highly conserved CArG box motifs (designated CB1 and CB2), and introduction of deletion mutations of either CB1 or CB2 reduced transcription in mesangial cells to near basal levels. Further analysis revealed that CB1 and CB2 acted synergistically when subcloned upstream of a heterologous, minimal thymidine kinase promoter. CB2 alone was sufficient to confer serum inducibility to a heterologous promoter, but both CB2 and CB1 were required for maximal levels of serum-induced transcription. Collectively, these results demonstrate that CB1 and CB2 cooperate to mediate serum-induced activation of the SM alpha-actin promoter in mesangial cells.
在体内，肾小球系膜细胞对肾小球损伤产生反应时会表达平滑肌α-肌动蛋白（SMα-肌动蛋白），并且SMα-肌动蛋白基因表达作为一种遗传标记，表征系膜细胞的活化表型。我们采用分子遗传学方法分析SMα-肌动蛋白启动子，并评估可能指导系膜细胞遗传转换为活化表型的转录机制。SMα-肌动蛋白启动子从-894到+1的序列可指导高水平转录，这种转录在血清限制的细胞中减弱，而在用血清或内皮素-1处理后上调。缺失分析揭示了一个核心启动子片段，从-122到+1位，该片段对于转录是必需且充分的。这种核心活性受到-670和-122之间上游序列的调节。122 bp的核心启动子包含两个高度保守的CArG框基序（分别命名为CB1和CB2），引入CB1或CB2的缺失突变会将系膜细胞中的转录降低到接近基础水平。进一步分析表明，当CB1和CB2亚克隆到异源最小胸苷激酶启动子上游时，它们协同发挥作用。单独的CB2足以赋予异源启动子血清诱导性，但最大水平的血清诱导转录需要CB2和CB1两者。总的来说，这些结果表明CB1和CB2协同介导血清诱导的系膜细胞中SMα-肌动蛋白启动子的活化。
