FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter

The insulin response element (IRE) in the IGFBP-1 promoter, and in other ge ne promoters, contains a T(A/G)TTT motif essential for insulin inhibition o f transcription. Studies presented here test whether FKHR may be the transc ription factor that confers insulin inhibition through this IRE motif. Immu noblots using antiserum to the synthetic peptide FKHR418-430 RNase protecti on, and Northerns blots show that FKHR is expressed in HEP G2 human hepatom a cells. Southwestern blots, electromobility shift assays, and DNase I prot ection assays show that Escherichia coli-expressed GST-FKHR binds specifica lly to IREs from the IGFBP-1, PEPCK and TAT genes; how-ever, unlike HNF3 be ta, another protein proposed to be the insulin regulated factor, GST-FKHR d oes not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR exp ression induced a 5-fold increase in activity of the native IGFBP-1 promote r but no increase in activity of promoter constructs containing insulin unr esponsive IRE mutants. These data suggest that FKHR, and/or a related famil y member, is the important T(G/A)TTT binding protein that confers the inhib itory effect of insulin on gene transcription.