Vanadate inhibits transcription of the rat insulin receptor gene via a proximal sequence of the 5′flanking region

Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5′flanking region of the IR gene (−1143/−252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at −455 and −396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the −1143/−252 fragment showed promoter activity. This was unaffected by deletion of the −1143/−761 sequence, but markedly decreased (90%) by additional deletion of the −760/−465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/−252, −760/−252 and −464/−252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 μM, and 70, 85 and 62% at 250 μM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position −464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.