Li J, Gao E, Mendelson C R
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038, USA.
J Biol Chem. 1998 Feb 20;273(8):4592-600. doi: 10.1074/jbc.273.8.4592.
Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form of PKA regulatory subunit RIalpha. We suggest that a PKA-induced increase of TTF-1 phosphorylation and TBE binding activity mediates cyclic AMP-induced expression of the SP-A gene in lung type II cells.
表面活性蛋白（SP）-A基因转录受增加环磷酸腺苷（cAMP）的因子刺激。在本研究中，我们观察到位于狒狒SP-A2（bSP-A2）基因5'端侧翼255个碱基对区域内的三个甲状腺转录因子-1（TTF-1）结合元件（TBEs）是cAMP最大程度诱导bSP-A2启动子活性所必需的。我们发现，在cAMP存在下培养的II型肺细胞的核提取物中，TTF-1的DNA结合活性增加。相比之下，对照和cAMP处理的II型细胞的核提取物中免疫反应性TTF-1蛋白的水平相似。cAMP处理也使免疫沉淀的TTF-1蛋白中[32P]正磷酸盐的掺入量显著增加。此外，用马铃薯酸性磷酸酶或碱性磷酸酶处理cAMP处理的II型细胞的核提取物，可消除cAMP诱导的TTF-1 DNA结合活性增加。有趣的是，已知能激活蛋白激酶C的佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）也增强了[32P]正磷酸盐掺入TTF-1蛋白；然而，TPA处理的II型细胞的核提取物中TTF-1的DNA结合活性降低。将编码TTF-1和蛋白激酶A催化亚基（PKA-cat）的表达载体与含有TBEs的SPA：人生长激素融合基因（来自狒狒SP-A2基因的5'侧翼DNA的255个碱基对与人生长激素相连，作为报告基因）或与含有三个串联TBEs且融合在bSP-A2基因TATA盒和转录起始位点上游的报告基因构建体一起共转染到A549肺腺癌细胞中。与在没有PKA-cat的情况下TTF-1的表达相比，TTF-1和PKA-cat的共表达使融合基因表达增加了3至4倍。此外，共转染PKA调节亚基RIalpha的显性负性形式可抑制TTF-1的转录活性。我们认为，PKA诱导的TTF-1磷酸化增加和TBE结合活性介导了II型肺细胞中cAMP诱导的SP-A基因表达。
