Mogayzel Peter J, Esmail Muneer, Wagner Traci L, Zeitlin Pamela L, Ashlock Melissa
Eudowood Division of Pediatric Respiratory Sciences, The Johns Hopkins Medical Institutions, 600 North Wolfe Street, Park 316, Baltimore, MD 21287-2533, USA.
Biochim Biophys Acta. 2002 Jul 19;1576(3):306-15. doi: 10.1016/s0167-4781(02)00395-0.
The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion. Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined. The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells. PMA treatment had no effect on luciferase activity in Calu-3 cells transiently transfected with plasmids containing luc driven by up to 2.3 kb of CFTR 5'-flanking DNA. PMA increased luciferase activity in transfected HT-29 cells. A more extensive region of DNA was evaluated using a yeast artificial chromosome (YAC) containing luc driven by approximately 335 of CFTR 5'-flanking DNA (y5'luc) stably introduced into HT-29 cells. Clonal cell lines containing y5'luc were created and assessed for luciferase activity at baseline and in response to forskolin and PMA. There was a wide range of baseline luciferase activities among the clones (42-1038 units/microg protein) that was not entirely due to the number of luc copies present within the cells. Treatment with both PMA and forskolin led to increased luciferase activity in six randomly selected clonal cell lines. As expected, endogenous CFTR expression increased in response to forskolin and decreased in response to PMA. These studies demonstrate that luc-containing YAC vectors can be used to study CFTR expression in human cells. In addition, these data suggest that important regulatory elements responsible for decreased CFTR expression in response to PMA are not located upstream of CFTR in the approximately 335 kb 5'-flanking sequence included in this YAC construct.
囊性纤维化跨膜传导调节因子基因（CFTR）以组织特异性和发育性方式受到调控。尽管一段时间以来已知佛波酯可降低CFTR mRNA水平较高的细胞系中CFTR的表达，但控制这种下调的顺式作用元件仍不清楚。使用转染到Calu-3和HT-29细胞中的含荧光素酶报告基因（luc）的质粒，评估CFTR最小启动子内顺式作用元件在调节对佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和福斯高林反应中的作用。用含有由长达2.3 kb的CFTR 5'侧翼DNA驱动的luc的质粒瞬时转染Calu-3细胞，PMA处理对荧光素酶活性没有影响。PMA增加了转染的HT-29细胞中的荧光素酶活性。使用含有由大约335 kb的CFTR 5'侧翼DNA（y5'luc）驱动的luc的酵母人工染色体（YAC）评估更广泛的DNA区域，该YAC稳定地导入HT-29细胞。创建了含有y5'luc的克隆细胞系，并在基线以及对福斯高林和PMA反应时评估荧光素酶活性。各克隆之间的基线荧光素酶活性范围很广（42 - 1038单位/微克蛋白质），这并不完全归因于细胞内存在的luc拷贝数。用PMA和福斯高林处理导致六个随机选择的克隆细胞系中的荧光素酶活性增加。如预期的那样，内源性CFTR表达对福斯高林反应增加，对PMA反应降低。这些研究表明，含luc的YAC载体可用于研究人细胞中CFTR的表达。此外，这些数据表明，在该YAC构建体包含的大约335 kb 5'侧翼序列中，负责PMA反应中CFTR表达降低的重要调控元件不在CFTR的上游。
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