In both 293-EcR and EcR-GR cells, ponasterone A induced transactivation was completely lost upon mutation of GRE #1 or the RRE (Figure 5B and 5C), with the firefly:renilla luciferase ratio being decreased by approximately10-fold on the RRE mutant construct compared to the mutant GRE#1 reporter (data not shown). These results suggest that both of these response elements serve as binding sites for the VgEcR-RXR transcriptional activation system. Transactivation was still observed with the mutated GRE #2 (Figure 5B and 5C) construct in both cell lines following ponasterone A treatment; however, it was slightly reduced compared to the non-mutated form. This indicates that this element may also be involved in activation of SEPP1 through VgEcR-RXR, although to a much lesser extent than GRE #1 or the RRE. The addition of dexamethasone plus ponasterone A resulted in attenuation of ponasterone A activity on the mutated GRE#2 luciferase reporter in the EcR-GR cells but not with the mutated GRE #1 reporter, indicating GRE #2 is not involved in the GR-mediated repression. FOXO1a has previously been shown to regulate SEPP1 transcription in hepatic cells
