Cloning the bovine Na+/myo-inositol cotransporter gene and characterization of an osmotic responsive promoter.
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.
Zhou
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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.
Cammarata
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D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA; D004848:Epithelium / Q000378:metabolism*; D005796:Genes / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics*; D006982:Hypertonic Solutions; D007908:Lens, Crystalline / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D014158:Transcription, Genetic / Q000235:genetics; D014882:Water-Electrolyte Balance / Q000235:genetics*
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D006982:Hypertonic Solutions; D008565:Membrane Proteins; D027981:Symporters; C073884:SLC5A3 protein, human; D004247:DNA; D012964:Sodium