Suwanichkul A, DePaolis L A, Lee P D, Powell D R
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1993 May 5;268(13):9730-6.
In hepatocytes, insulin-like growth factor-binding protein-1 (IGFBP-1) levels are increased by glucocorticoids and by agents that raise intracellular cAMP levels such as glucagon, theophylline, forskolin, and cAMP analogues. In contrast, insulin lowers IGFBP-1 levels, an effect dominant over the glucocorticoid and cAMP effects. Previous studies showed that dibutyryl cAMP (Bt2cAMP) and theophylline increase IGFBP-1 promoter activity in HEP G2 human hepatoma cells and that insulin abolishes this increase. In studies reported here, HEP G2 cells were used to further evaluate the role of cAMP in stimulating IGFBP-1 expression. Initial studies found that either 0.5 or 5.0 mM Bt2cAMP alone, or the combination of 0.5 mM Bt2cAMP and 2 mM theophylline, increased IGFBP-1 protein levels, mRNA levels, and promoter activity, but that the addition of theophylline to Bt2cAMP was required to give a approximately 5-fold increase in promoter activity. Deletion mutations of the IGFBP-1 promoter were used to show that much of the effect of Bt2cAMP and theophylline was conferred by the region between 269 and 246 base pairs (bp) 5' of the IGFBP-1 mRNA cap site. DNase I protection assays showed that HEP G2 nuclear extract footprinted the region from 273 to 249 bp 5' of the cap site; this region, designated P2, has a central CGTCA motif common to cAMP-responsive elements (CREs). Mutating the CGTCA motif in the 1205-bp IGFBP-1 promoter construct to TAGCA led to a 51% decrease in the ability of Bt2cAMP and theophylline to stimulate IGFBP-1 promoter activity above control levels. In addition, cotransfection of the catalytic subunit of cAMP-dependent protein kinase A (PKA) with the native 1205-bp IGFBP-1 promoter construct stimulated IGFBP-1 promoter activity 3.9-fold, but the TAGCA mutation decreased by 73% the ability of PKA to stimulate IGFBP-1 promoter activity above control levels. Mutating the CGTCA motif to TAGCA also blocked the ability of both crude HEP G2 nuclear extract and recombinant CRE-binding protein to bind to the P2 element. These data suggest that the P2 element is a CRE that confers at least part of the stimulatory effect of cAMP on the human IGFBP-1 promoter.
在肝细胞中，糖皮质激素以及能提高细胞内cAMP水平的物质（如胰高血糖素、茶碱、福斯可林和cAMP类似物）可使胰岛素样生长因子结合蛋白-1（IGFBP-1）水平升高。相反，胰岛素可降低IGFBP-1水平，此效应强于糖皮质激素和cAMP的作用。既往研究表明，二丁酰cAMP（Bt2cAMP）和茶碱可增加人肝癌细胞系HEP G2中IGFBP-1启动子活性，而胰岛素可消除这种增加。在本文报道的研究中，利用HEP G2细胞进一步评估cAMP在刺激IGFBP-1表达中的作用。初步研究发现，单独使用0.5 mM或5.0 mM Bt2cAMP，或0.5 mM Bt2cAMP与2 mM茶碱联合使用，均可增加IGFBP-1蛋白水平、mRNA水平及启动子活性，但将茶碱添加到Bt2cAMP中可使启动子活性增加约5倍。对IGFBP-1启动子进行缺失突变表明，Bt2cAMP和茶碱的大部分作用是由IGFBP-1 mRNA帽位点5'端269至246碱基对（bp）之间的区域介导的。DNase I保护试验表明，HEP G2细胞核提取物可使帽位点5'端273至249 bp区域出现足迹；该区域命名为P2，具有cAMP反应元件（CRE）共有的核心CGTCA基序。将1205 bp的IGFBP-1启动子构建体中的CGTCA基序突变为TAGCA，可使Bt2cAMP和茶碱刺激IGFBP-1启动子活性高于对照水平的能力降低51%。此外，将cAMP依赖性蛋白激酶A（PKA）的催化亚基与天然的1205 bp IGFBP-1启动子构建体共转染，可使IGFBP-1启动子活性增加3.9倍，但TAGCA突变使PKA刺激IGFBP-1启动子活性高于对照水平的能力降低73%。将CGTCA基序突变为TAGCA也可阻断粗制HEP G2细胞核提取物和重组CRE结合蛋白与P2元件结合的能力。这些数据表明，P2元件是一种CRE，它赋予cAMP对人IGFBP-1启动子至少部分刺激作用。
