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

Abstract

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.

Keywords: C/EBPβ, C/CAAT/enhancer binding protein β; FoxO1, Forkhead box protein O1; Gene transcription; HMGA1, high mobility group A1 protein; HNF4, hepatocyte nuclear factor 4; Hepatoma cells; IGFBP-1, insulin-like growth factor binding protein 1; IR, insulin receptor; Insulin receptor; Liver; PEPCK, phosphoenolpyruvate carboxykinase; PI3K, phosphatidyl inositol 3-kinase; Rat; SINE, short interspersed nuclear element; STZ, streptozotocin; Sp1, specificity protein 1; TCF7L2, T-cell specific transcription factor 7-like 2; Vanadate.

Fig. 1

Localization of transcriptional start sites in the 5′flanking region of the rat insulin receptor gene by RNase protection analysis. The −464/−252 and −760/−252 IR gene fragments generated from the −1143/−252 parental fragment by Pvu2 and Dra1, respectively, were used as templates to synthesize two [P³²] labeled antisense RNA probes. Following incubation of these probes with 40 μg of total liver RNA, nonhybridized probes were digested by a mixture of RNase T1 and RNase A, and hybrids were analyzed by electrophoresis on 6% polyacrylamide gels containing 6M urea and radioautography. The size of the protected RNA fragments (arrows on the left) was determined using a dideoxy sequencing ladder run in parallel on the same gels (shown on the right).

Fig. 2

Promoter activity of rat insulin receptor gene 5′flanking sequences in untreated and vanadate-treated Fao hepatoma cells. Fao hepatoma cells were transfected with the indicated IR gene fragment fused to the pGL3 plasmid as described in Materials and Methods. Transfected cells were left untreated or treated with vanadate at the indicated concentration for 24 h. Following cell lysis, lysates were clarified and assayed for luciferase activity with results normalized to protein and expressed relatively to empty pGL3 activity. For each of the three IR-luciferase constructs, asterisks indicate a statistically significant difference between untreated and vanadate-treated cells.

Fig. 3

Schematic representation of the 5′flanking −1143/−252 sequence of the rat IR gene. This figure shows the conserved sequence (−523/−407) (orange box), the transcription start sites (arrows), and the binding sites of transcription factors potentially involved in IR gene transcription in untreated and vanadate-treated hepatoma cells (colored ellipses and triangles).