McDonald R A, Matthews R P, Idzerda R L, McKnight G S
Department of Pharmacology, University of Washington, Seattle 98195, USA.
Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7560-4. doi: 10.1073/pnas.92.16.7560.
The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.
囊性纤维化跨膜传导调节因子（CFTR）作为一种氯离子通道，在被环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）磷酸化后被激活。我们证明，PKA在维持人结肠癌细胞系T84中CFTR基因的基础表达方面也起着关键作用。通过表达显性负性调节亚基或用PKA选择性抑制剂N-[2-(对溴肉桂酰胺基)乙基]-5-异喹啉磺酰胺（H-89）处理来抑制PKA活性，会导致CFTR基因表达完全被抑制，而不影响其他组成型活性基因。与荧光素酶报告基因（CFTR-luc）相连的CFTR启动子2.2 kb区域的基础表达对PKA表现出相同的依赖性。cAMP诱导CFTR超过基础水平的能力具有细胞类型特异性。在T84细胞中，内源性CFTR基因和CFTR-luc都仅表现出适度的诱导性（约2倍），而在人绒毛膜癌细胞系JEG-3中，CFTR-luc可被诱导至少4倍。在CFTR启动子的-48至-41位存在一个变异的cAMP反应元件，该序列的突变会阻断基础表达。我们得出结论，通过PKA起作用的cAMP是CFTR基因基础表达的重要调节因子，并且可能在反应性细胞类型中介导CFTR的诱导。
