Figure 8.

hCAR downregulates EPO expression through suppression of HNF4α.A, RT–PCR was used to analyze expression of hCAR, EPO, HNF4α, and HIF1β in HepG2-hCAR and Hep3B-hCAR cells 72 h after treatment with vehicle control or Dox (1 μg/ml) as detailed in the “Experimental procedures” section. B, the protein levels of HNF4α were analyzed by immunoblotting in HepG2-hCAR, Hep3B-hCAR, as well as normal HepG2 and Hep3B cells treated with vehicle control or Dox for 72 h. Relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. C, knockdown of HNF4α expression in HepG2 and Hep3B cells via lentivirus shRNA targeting different regions of HNF4α (shHNF4α-1 and shHNF4α-2) resulted in downregulation of EPO expression in these cells. D, overexpression of HNF4α partially rescues hCAR-mediated downregulation of EPO expression in HepG2-hCAR and Hep3B-hCAR stable cell lines. E, EMSA was performed to detect interaction between the EPO–DR2 motif and nuclear extract samples isolated from HepG2-hCAR and Hep3B-hCAR cells treated with vehicle control or Dox for 72 h. HNF4α antibody was used for measuring specific supershift of the HNF4α/DR2 band. 1: NE of HepG2-hCAR-control; 2: NE of HepG2-hCAR-Dox; 3: NE of Hep3B-hCAR-control; and 4: NE of Hep3B-hCAR-Dox. F, HEK293T cells were transfected with EPO-3′UTR-enhancer reporter construct in the presence of HNF4α and/or hCAR expression vectors as indicated, and luciferase activities were determined 48 h after transfection. Three independent measures from each group were analyzed and expressed as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; DR2, direct repeat 2; EPO, erythropoietin; hCAR, human constitutive androstane receptor; HEK293T, human embryonic kidney 293T cell line; HIF1β, hypoxia-inducible factor 1β; HNF4α, hepatocyte nuclear factor 4 alpha.