Zhou C, Cammarata P R
Department of Anatomy and Cell Biology, University of North Texas Health Science Center at Fort Worth/North Texas Eye Research Institute, Fort Worth, Texas 76107, USA.
Exp Eye Res. 1997 Sep;65(3):349-63. doi: 10.1006/exer.1997.0335.
Hyperosmotic-induced enhancement of myo-inositol accumulation in cultured bovine lens epithelial cells stems from increased uptake activity due to upregulation of Na+/myo-inositol cotransporter mRNA and de novo synthesis in myo-inositol carrier protein. The molecular mechanism of osmoregulation of Na+/myo-inositol cotransporter gene transcription was further investigated. The effect of hypertonicity on transcription initiation of the Na+/myo-inositol cotransporter gene was examined by use of the rapid amplification of cDNA ends (RACE) technique. 5"-RACE analysis revealed that one hypertonic and two isotonic (i.e. physiologic) transcription start sites are present in the Na+/myo-inositol cotransporter gene of cultured bovine lens epithelial cells. Moreover, the bovine Na+/myo-inositol cotransporter gene was cloned and its promoters were characterized by transient transfection assay using luciferase reporter constructs of various fragments of the 5"-flanking regions upstream of the individual transcription start sites. Among its promoters, only one was osmotically responsive and showed approximately a 3-fold induction of activity subsequent to hypertonic insult. Transient transfection assays revealed that the region between -398 and -331 bp, upstream of this hypertonic transcription start site, contains the putative osmotic response element. Preferential utilization of this osmotically responsive promoter contributes to the elevation of Na+/MI cotransporter mRNA abundance and increased myo-inositol uptake activity as initiated by osmotic stress.
高渗诱导培养的牛晶状体上皮细胞中肌醇积累增强，源于钠/肌醇共转运蛋白mRNA上调和肌醇载体蛋白从头合成导致的摄取活性增加。进一步研究了钠/肌醇共转运蛋白基因转录的渗透调节分子机制。利用cDNA末端快速扩增（RACE）技术检测了高渗对钠/肌醇共转运蛋白基因转录起始的影响。5'-RACE分析显示，培养的牛晶状体上皮细胞的钠/肌醇共转运蛋白基因中存在一个高渗转录起始位点和两个等渗（即生理）转录起始位点。此外，克隆了牛钠/肌醇共转运蛋白基因，并使用各个转录起始位点上游5'-侧翼区域不同片段的荧光素酶报告构建体通过瞬时转染试验对其启动子进行了表征。在其启动子中，只有一个对渗透压有反应，在高渗损伤后活性约有3倍的诱导。瞬时转染试验表明，该高渗转录起始位点上游-398至-331 bp之间的区域包含假定的渗透反应元件。这种对渗透压有反应的启动子的优先利用有助于钠/肌醇共转运蛋白mRNA丰度的升高和渗透压应激引发的肌醇摄取活性增加。
