Estrogen response element-independent regulation of gene expression by genistein in intestinal cells

Isoflavone, genistein, was shown to have antioxidant and antitumor activities. We have reported the stimulatory effect of genistein on the expression of antioxidant and metal-binding protein, metallothionein IIA (MTIIA), in human intestinal Caco-2 cells. Genistein has been shown to up-regulate the expression of other genes through estrogen response element (ERE) but the ERE sequence is not in the MTIIA promoter. In this paper, we investigated the ERE-independent mechanism that mediates the stimulatory effect of genistein. Genistein enhanced the expression of human MTIIA promoter (up to -426)-containing reporter genes, thus supporting a promoter-specific transcriptional regulation. A shorter MTIIA promoter (-83 to +27) was found to be able to mediate the full reporter gene response to genistein in a dose- and time-dependent fashion. Further deletion and mutation analysis revealed that the GC-rich Sp1 binding sequence was the target of the stimulation. Genistein was known to bind to estrogen receptors (ER). When cells were cotransfected with ERβ, the stimulatory effect of genistein on the reporter gene containing the GC-rich promoter sequence increased further and a similar result was observed for breast cancer MCF-7 cells. Inhibitors of protein kinase A could block the response to genistein but the phosphorylation of Sp1 protein per se was not affected by the genistein treatment. Our observation could help to evaluate the biological significance of genistein, which is used widely as a supplement.