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

Peroxisome proliferator-activated receptor-alpha (PPAR alpha) is a member of the nuclear receptor superfamily involved in hepatocarcinogenesis in rodents. In previous studies on liver tumor tissues, PPAR alpha mRNA expression was found to be significantly higher and overexpression of ER alpha inhibited the PPAR alpha expression, cell-proliferation and also induced apoptosis in Hep3B cell. However, the role of ER beta is not known yet. Therefore, the aim of this study is to define the role of ER beta on PPAR alpha in Hep3B cells. The effect of PPAR alpha signaling cascade were monitored by inducing Hep3B cells by fenofibrate. Further the cells were transfected with pCMV-ER beta and the consequences of ER beta-overexpression on the PPAR alpha 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 ER beta modulates PPAR alpha expression by binding to PPAR alpha promoter region to repress PPAR alpha promoter activity. In addition, the direct interaction between ER beta and PPAR alpha proteins was verified by co-immunoprecipitation assay. Our results show that the overexpressed ER beta 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 E-2/ER beta resulted in the enhancement of mitochondria dependent apoptotic pathway and the attenuation of cell proliferation. Moreover, the overexpressed ER beta reduced the mRNA and protein levels of PPAR alpha and its downstream Acyl-CoA oxidase (ACO). EMSA results show that ER beta directly binds to PPRE and inhibit PPAR alpha gene expression and according to immunoprecipitation assay ER beta also binds strongly with PPAR alpha. The E-2/ER beta further inhibited the fenofibrate-induced nuclear translocation of PPAR alpha. Taken together, ER beta might directly downregulate PPAR alpha gene expression and inhibit the nuclear translocation to suppress the proliferation and induce the apoptosis of Hep3B cells.