Characterization of the human liver fructose-1,6-bisphosphatase gene promoter

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogen ic enzyme, catalyses the hydrolysis of fructose 1,6-bisphosphate to fructos e 6-phosphate and P-1. Enzyme activity is mainly regulated by the allosteri c inhibitors fructose, 2,6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase pr omoter has not been studied in detail. Here we report an in vitro character ization of the FBPase promoter with respect to the elements that are requir ed for basal promoter activity. Transient transfection of H4IIE rat hepatom a cells, combined with site-directed mutagenesis, demonstrated that an enha ncer box, three GC-boxes and a nuclear factor kappaB (NF-kappaB)-binding el ement are important for hepatic FBPase promoter activity. These elements ar e found in the region located between -405 to +25 bp relative to the transc ription start site. Electrophoretic-mobility-shift assays and supershift an alysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificit y protein 1 (Sp1)/Sp3 and NF-kappaB respectively bind to these sites. The p resent study provides the basis for a more comprehensive screening for muta tions in FBPase-deficient patients and for further studies of the transcrip tional regulation of this gene.