E₂/ERβ Inhibits PPARα to Regulate Cell-Proliferation and Enhance Apoptosis in Hep3B-Hepatocellular Carcinoma

Peroxisome proliferator-activated receptor-α (PPARα) is a member of the nuclear receptor superfamily involved in hepatocarcinogenesis in rodents. In previous studies on liver tumor tissues, PPARα mRNA expression was found to be significantly higher and overexpression of ERα inhibited the PPARα expression, cell-proliferation and also induced apoptosis in Hep3B cell. However, the role of ERβ is not known yet. Therefore, the aim of this study is to define the role of ERβ on PPARα in Hep3B cells. The effect of PPARα signaling cascade were monitored by inducing Hep3B cells by fenofibrate. Further the cells were transfected with pCMV-ERβ and the consequences of ERβ-overexpression on the PPARα induced changes such as enhanced cell-proliferation and suppressed apoptosis were determined using western blot analysis and TUNEL assay. The EMSA was used to identify whether ERβ modulates PPARα expression by binding to PPARα promoter region to repress PPARα promoter activity. In addition, the direct interaction between ERβ and PPARα proteins was verified by co-immunoprecipitation assay. Our results show that the overexpressed ERβ not only attenuated the effects of fenofibrate to induce the levels of apoptosis protein such as Cyt.c, Caspase 9 and Caspase 3 but also inhibited the levels of survival protein such Bcl-xL, p-Bad, cyclin A and cyclin E. All these effects of E₂/ERβ resulted in the enhancement of mitochondria dependent apoptotic pathway and the attenuation of cell proliferation. Moreover, the overexpressed ERβ reduced the mRNA and protein levels of PPARα and its downstream Acyl-CoA oxidase (ACO). EMSA results show that ERβ directly binds to PPRE and inhibit PPARα gene expression and according to immunoprecipitation assay ERβ also binds strongly with PPARα. The E₂/ERβ further inhibited the fenofibrate-induced nuclear translocation of PPARα. Taken together, ERβ might directly downregulate PPARα gene expression and inhibit the nuclear translocation to suppress the proliferation and induce the apoptosis of Hep3B cells.