Differential activation of the IGF binding protein-3 promoter by butyrate in prostate cancer cells

Abstract

Sodium butyrate (NAB), a dietary micronutrient, is a potent growth inhibitor that initiates cell differentiation in many cell types, including prostate cancer cells. The molecular mechanisms by which these effects occur remain largely unknown. In this study, we investigated the effects of NaB on the expression of IGF binding protein (IGFBP)-3, a known growth regulator, in two human prostate cancer cell lines (PC-3 and LNCaP). Treatment with NaB (0-10 mM) caused a dose-dependent stimulation of IGFBP-3 mRNA expression and parallel increases in protein levels. A specific histone deacetylase inhibitor, trichostatin A (TSA) similarly induced IGFBP-3 expression, indicating that histone hyperacetylation may be critical in the regulation of IGFBP-3 expression. To investigate the molecular mechanism of NaB-regulated IGFBP-3 expression, 1.87 kb of the human IGFBP-3 gene promoter was cloned into the pGL2-basic luciferase reporter vector. In both PC-3 and LNCaP cells, NaB (10 mM) significantly increased luciferase activity 20- to 30-fold, compared with the untreated control. However, using 5′ sequential deletion constructs of the IGFBP-3 promoter, the NaB response sequences in the IGFBP-3 promoter were different in PC-3 and LNCaP cells. Our studies identified a region, -75 to +69 from the start of transcription (+1), that is fully inducible by NaB treatment in LNCaP cells, but not in PC-3 cells. Unlike other well characterized NaB-regulated genes, Sp1 DNA sequences are not involved in NaB up-regulation of IGFBP-3 gene in LNCaP cells. Further deletion studies identified two independent regions critical for NaB-induced transactivation in LNCaP cells. These regions contain consensus binding sites for p53 and GATA, respectively, but mutational analyses and gel shift assays suggested that, while the p53 response element is required for NaB responsiveness, neither p53 nor GATA are involved. In summary, we have demonstrated that 1) NaB significantly up-regulates IGFBP-3 mRNA and protein levels in PC-3 and LNCaP prostate cancer cells; and 2) novel butyrate-responsive elements lacking consensus Sp1 sites are used in LNCaP cells.