Expression of metallothoinein isoform 3 is restricted at the post-transcriptional level in human bladder epithelial cells.
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.
Garrett
Scott H
SH
Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202, USA.
Park
Seongmi
S
Sens
Mary Ann
MA
Somji
Seema
S
Singh
Rajendra K
RK
Namburi
Venugopal B R K
VB
Sens
Donald A
DA
D002104:Cadmium / Q000633:toxicity; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008668:Metallothionein / Q000032:analysis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic; D014162:Transfection; D001743:Urinary Bladder / Q000378:metabolism*
D020033:Protein Isoforms; D012333:RNA, Messenger; D002104:Cadmium; D008668:Metallothionein