Garrett Scott H, Park Seongmi, Sens Mary Ann, Somji Seema, Singh Rajendra K, Namburi Venugopal B R K, Sens Donald A
Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202, USA.
Toxicol Sci. 2005 Sep;87(1):66-74. doi: 10.1093/toxsci/kfi231. Epub 2005 Jun 15.
This study was designed to define the effect that overexpression of MT-3 would have on a cell culture model of bladder urothelium. Stable and inducible transfection was used to achieve overexpression of the MT-3 gene in the UROtsa cell line. When the UROtsa cells were stably transfected with the MT-3 coding sequence, there was highly elevated expression of MT-3 mRNA, but no MT-3 protein. An inducible vector showed that low basal levels of MT-3 mRNA and protein could be produced, but that induction only increased MT-3 mRNA and not protein. The clones expressing low basal levels of MT-3 protein also had reduced growth rates compared to control cells. Site directed mutagenesis was used to produce an MT-3 coding sequence where the prolines in positions 7 and 9 were converted to threonines. When this altered MT-3 was stably transfected into the UROtsa cells, the cells were able to accumulate the mutated form of the MT-3 protein. These studies show that MT-3 protein expression is inhibited by post-transcriptional control in the urothelial cell. Modifying the MT-3 protein to resemble the MT-1 isoform removes this component of post-transcriptional control and allows accumulation of the mutated MT-3 protein. The altered sequence involved in post-transcriptional control of MT-3 protein expression is the same sequence implicated in the neuronal growth inhibitory activity associated specifically with the MT-3 isoform of the MT gene family.
本研究旨在确定MT-3过表达对膀胱尿路上皮细胞培养模型的影响。采用稳定和诱导转染的方法在UROtsa细胞系中实现MT-3基因的过表达。当用MT-3编码序列稳定转染UROtsa细胞时，MT-3 mRNA表达显著升高，但未检测到MT-3蛋白。诱导型载体显示可产生低基础水平的MT-3 mRNA和蛋白，但诱导仅增加MT-3 mRNA而非蛋白。与对照细胞相比，表达低基础水平MT-3蛋白的克隆生长速率也降低。使用定点诱变产生MT-3编码序列，其中第7位和第9位的脯氨酸被转换为苏氨酸。当将这种改变的MT-3稳定转染到UROtsa细胞中时，细胞能够积累突变形式的MT-3蛋白。这些研究表明，尿路上皮细胞中MT-3蛋白的表达受到转录后调控的抑制。将MT-3蛋白修饰为类似于MT-1同工型可消除转录后调控的这一成分，并允许突变的MT-3蛋白积累。参与MT-3蛋白表达转录后调控的改变序列与MT基因家族MT-3同工型特有的神经元生长抑制活性相关的序列相同。
