CYP1B1 Activates Wnt/beta-Catenin Signaling through Suppression of Herc5-Mediated ISGylation for Protein Degradation on beta-Catenin in HeLa Cells

Abstract

Cytochrome P450 1B1 (CYP1B1) acts as a hydroxylase for estrogen and activates potential carcinogens. Moreover, its expression in tumor tissues is much higher than that in normal tissues. Despite this association between CYP1B1 and cancer, the detailed molecular mechanism of CYP1B1 on cancer progression in HeLa cells remains unknown. Previous reports indicated that the mRNA expression level of Herc5, an E3 ligase for ISGylation, is promoted by CYP1B1 suppression using specific small interfering RNA, and that ISGylation may be involved in ubiquitination related to beta-catenin degradation. With this background, we investigated the relationships among CYP1B1, Herc5, and beta-catenin. RT-PCR and western blot analyses showed that CYP1B1 overexpression induced and CYP1B1 inhibition reduced, respectively, the expression of Wnt/beta-catenin signaling target genes including beta-catenin and cyclin D1. Moreover, HeLa cells were treated with the CYP1B1 inducer 7,12-dimethylbenz[a] anthracene (DMBA) or the CYP1B1 specific inhibitor, tetramethoxystilbene (TMS) and consequently DMBA increased and TMS decreased beta-catenin and cyclin D1 expression, respectively. To determine the correlation between CYP1B1 expression and ISGylation, the expression of ISG15, a ubiquitin-like protein, was detected following CYP1B1 regulation, which revealed that CYP1B1 may inhibit ISGylation through suppression of ISG15 expression. In addition, the mRNA and protein expression levels of Herc5 were strongly suppressed by CYP1B1. Finally, an immunoprecipitation assay revealed a direct physical interaction between Herc5 and beta-catenin in HeLa cells. In conclusion, these data suggest that CYP1B1 may activate Wnt/beta-catenin signaling through stabilization of beta-catenin protein from Herc5-mediated ISGylation for proteosomal degradation.