Transcriptional regulation of chicken CXR, FXR, LXR and PPARα in LMH cells by glucocorticoids. LMH cells were treated for 16 hours with vehicle or 50 μM dexamethasone before cells were lysed and RNA was analysed by Northern blotting with probes for CXR, chicken PPARα, chicken LXR, chicken FXR or chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Phylogeny of xenobiotic-sensing nuclear receptors from different species. A, A non-rooted phylogenetic tree depicts the relationship between mammalian CARs and PXRs and non-mammalian intermediate receptors. The scale bar represents 0.1 amino acid substitutions per site. B, The sites of intron-exon junctions in the coding regions of CXR, PXR and CAR are highly conserved as depicted in an alignment of the amino acid sequences of these receptors.

Nuclear CXR protein levels decrease after PB-treatment. LMH cells were treated with vehicle or 400 μM PB for 16 hours before cells were lysed and CXR protein levels in the total lysate (lanes 1 and 2) and in nuclear extracts (lanes 3 and 4) determined by Western blot. As controls, CV-1 cells were transfected with control vector or CXR expression vector (lanes 5 and 6). MW, molecular weight in kDa.

Reduced drug-induction of drug-responsive enhancer elements from CYP2H1, CYP3A37, CYP2C45 and ALAS1 in LMH cells stably expressing CXR RNAi. A, mRNA levels of endogenous CXR in LMH cells expressing pSUPER expression vector or CXR RNAi. CXR levels were measured by real-time PCR in LMH cells that stably express control vector or CXR RNAi. B–E, Phenobarbital-induction of drug-responsive enhancer elements from CYP2H1 (B), CYP3A37 (C), CYP2C45 (D) and ALAS1 (E) in LMH cells expressing pSUPER or CXR RNAi. LMH cells were transfected with the reporter gene plasmids and subsequently treated with vehicle or 400 μM PB for 16 hours before reporter gene levels were determined.

Cellular localization of CXR in transiently transfected LMH cells. A, Full-length CXR, CXR with GFP attached at its C-terminus or at its N-terminus or N-terminal GFP-CXR fusion protein mutated in its xenochemical response signal (XRS) at positions 356, 359 and 362 were expressed in CV-1 cells together with a reporter plasmid containing the CYP2H1 264-bp PBRU. After transfection, the cells were treated with either vehicle, 400 μM PB or 10 μM clotrimazole before cells were lysed and analysed for reporter gene expression. Values are the average of three independent experiments and error bars represent standard deviations. B–F, LMH cells were transfected with either pEGFP-vector alone (B), expression vector for N-terminal GFP-CXR fusion protein treated with vehicle (C), 400 μM PB (D) or 0.1 μM okadaic acid (E) for 16 hours or GFP-CXR fusion protein with the xenochemical response signal mutation as described in Fig. 4A (F). Cells were stained with 300 nM DAPI in PBS and analysed for DAPI and GFP-specific light emissions at 461 nm and 507 nm using excitation wavelengths of 358 nm and 488 nm, respectively. Size bars stand for 20 μm.

Drug-induction of the 264-bp PBRU is abolished by a dominant-negative CXR allele. A, CXR was subcloned into the pHook-2 expression plasmid (Hook-2) either full-length CXR in positive orientation (CXR+), negative orientation (CXR-), lacking its N-terminal amino acids 1–29 (ΔN-term), full-length CXR with four of its cysteine residues (cysteine 31, 34, 83 and 86) in the DNA-binding domain mutated (DBD) or lacking its C-terminal amino acids 383–391 containing the activation function AF-2 (ΔAF2). B, Electrophoretic mobility shift assays with mock in vitro transcribed/translated reticulocyte lysate (lane 1), expression plasmid pHook-2 (lane 2) and either expression plasmids for the different CXR alleles alone (lanes 3–7) or together with a pSG5-expression plasmid for chicken RXRγ (lanes 8–12). The arrow indicates the specific shift of CXR/RXR complexes with the radiolabeled CYP2H1 264-bp PBRU. C, pHook-2 expression plasmids without insert or containing the various CXR alleles were co-transfected with the CYP2H1 264-bp PBRU in the pBLCAT5 reporter vector as well as a lacZ-expression vector for normalization of transfection efficiencies into non-drug responsive CV-1 cells. After transfection, the cells were treated with either vehicle or 400 μM PB for 24 hours before cells were lysed and analysed for reporter gene expression and β-galactosidase expression. Values are the average of the relative CAT expression normalized for β-galactosidase levels of three independent experiments and error bars represent the standard deviation. D, pHook-2 expression plasmids without insert or containing the various CXR alleles were co-transfected with the CYP2H1 264-bp PBRU in the pBLCAT5 reporter vector into drug-responsive LMH cells expressing endogenous CXR. After transfection, the cells were treated with either vehicle or 400 μM PB for 24 hours before cells were lysed and analysed for reporter gene expression and β-galactosidase expression. Values are the average of the relative CAT expression normalized for β-galactosidase levels of three independent experiments and error bars represent the standard deviation.