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.