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. 2007;35(7):2177-90.
doi: 10.1093/nar/gkm090. Epub 2007 Mar 13.

Transactivation of a DR-1 PPRE by a Human Constitutive Androstane Receptor Variant Expressed From Internal Protein Translation Start Sites

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Free PMC article

Transactivation of a DR-1 PPRE by a Human Constitutive Androstane Receptor Variant Expressed From Internal Protein Translation Start Sites

Matthew A Stoner et al. Nucleic Acids Res. .
Free PMC article

Abstract

Downstream in-frame start codons produce amino-terminal-truncated human constitutive androstane receptor protein isoforms (DeltaNCARs). The DeltaNCARs are expressed in liver and in vitro cell systems following translation from in-frame methionine AUG start codons at positions 76, 80, 125, 128, 168 and 265 within the full-length CAR mRNA. The resulting CAR proteins lack the N-terminal DNA-binding domain (DBD) of the receptor, yielding DeltaNCAR variants with unique biological function. Although the DeltaNCARs maintain full retinoid X receptor alpha (RXRalpha) heterodimerization capacity, the DeltaNCARs are inactive on classical CAR-inducible direct repeat (DR)-4 elements, yet efficiently transactivate a DR-1 element derived from the endogenous PPAR-inducible acyl-CoA oxidase gene promoter. RXRalpha heterodimerization with CAR1, CAR76 and CAR80 isoforms is necessary for the DR-1 PPRE activation, a function that exhibits absolute dependence on both the respective RXRalpha DBD and CAR activation (AF)-2 domains, but not the AF-1 or AF-2 domain of RXRalpha, nor CAR's DBD. A new model of CAR DBD-independent transactivation is proposed, such that in the context of a DR-1 peroxisome proliferator-activated response element, only the RXRalpha portion of the CAR-RXRalpha heterodimer binds directly to DNA, with the AF-2 domain of tethered CAR mediating transcriptional activation of the receptor complex.

Figures

Figure 1.
Figure 1.
Western immunoblot analyses. (A) Expression of CAR-variant proteins and RXRα protein in human primary hepatocytes from four donors. Lanes 1–4, 150 μg whole-cell protein extract from each of four donors; lanes 5 and 6 are positive control whole-cell protein extracts from HEK-293T human embryonic kidney cell lines transfected with expression plasmids for CAR 1 or CAR 76 and treated with 1 μM MG-132 proteasome inhibitor to augment CAR expression. Ponceau S staining was performed to indicate total protein loading in each lane. (B) CAR protein expression in COS-1 cells transiently transfected with CAR and CAR splice variant expression plasmids. Lane 1, empty vector transfection (V); lane 2, CAR wild-type (WT); lane 3, CAR4aa (SPTV amino acids inserted between exons 6/7); lane 4, CAR5aa (APYLT amino acids inserted between exons 7/8); CAR-DBL (four and five amino acid insertion); and CARΔ7 (partial deletion of exon 7 coding for 39 amino acids). Embedded asterisks denote unexpected lower molecular weight CAR products.
Figure 2.
Figure 2.
ClustalW alignment of human, mouse and rat CAR mRNA sequences. (A) Numbers above sequences denote NetStart1.0-predicted in-frame internal protein translation start sites. Numbers above sequences represent codon numbering based on human sequence. Numbers listed to the side indicate relative position of nucleotides within coding sequence, asterisks denote sequence conservation across species. (B) CAR protein and mRNA sequences alignment. Predicted CAR translation start sites are denoted by arrows at 1, 76, 80, 125, 128, 168 and 265. For full-length CAR protein, translation begins from a start codon 1 in exon 2 and continues to codon 348 in exon 9.
Figure 3.
Figure 3.
Western immunoblot assay. (A) CAR full-length protein and translational variants are expressed in Adv-CAR-infected human hepatocytes and in in vitro wheat germ expression system. Lane 1, human primary hepatocytes infected with adenovirus expressing wild-type CAR mRNA; (lanes 2–4, wheat germ lysates); lane 2, empty vector control; lane 3, pCDNA3.1(−)CAR 1; lane 4, pCDNA3.1(−)CAR M1K—start methionine mutated to lysine. Numbers to the left of figure indicate predicted internal translation variants' relative molecular weights (Mr). (B) CAR 1 and CAR M1K differentially transactivate DR-4 and DR-1 elements. COS-1 cells were transiently transfected with 100 ng of reporters p(DR-4)3-tk-Luc, p(rCRBPII)-tk-Luc or p(rAox-PPRE)4-tk-Luc, 50 ng RXRα and CAR expression plasmids and 10 ng pRL-CMV.
Figure 4.
Figure 4.
Truncated forms of CAR are expressed and activate transcription through a special DR-1 element. (A) SDS-PAGE of coupled in vitro transcription/translation of [35S]-methionine-labeled CAR in wheat germ lysate (lanes 1–5) and rabbit reticulocyte lysate (lanes 6–10). Lanes 1 and 6, pCDNA3.1(−)CAR 1; lanes 2 and 7, pCDNA3.1(−)CAR M1K; lanes 3 and 8, pCDNA3.1(−)CAR 76; lanes 4 and 9, pCDNA3.1(−)CAR 1, M76K; and lanes 5 and 10, pCDNA3.1(−)CAR 1, M76K/M80K. (B) Differential activation of p(DR-4)3-tk-Luc and p(rAox-PPRE)4-tk-Luc by amino-terminally truncated forms of CAR. (C) Activation of p(rAox-1198/-463)-tk-Luc, a genomic promoter fragment containing a single copy of the PPRE element, by CAR 1 and CAR 76. COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(−) empty vector or pCDNA3.1(−)CAR plasmids, 50 ng pCDNA3.1(+)RXRα and 10 ng pRL-CMV (for transfection normalization) and assayed for luciferase activity 24–48 h post-transfection.
Figure 5.
Figure 5.
Transient transfection assays. (A) An intact DNA-binding domain is necessary for CAR transcriptional activity on a DR-4 element. COS-1 cells were transfected with 100 ng p(DR-4)3-tk-Luc, 50 ng empty vector or RXRα, 50 ng empty vector or CAR constructs and 10 ng pRL-CMV. (B) CAR amino acids 103–348 and co-transfected RXRα are required for full transcriptional activity on a DR-1 element. COS-1 cells were transfected with 100 ng p(rAox-PPRE)4-tk-Luc, 50 ng empty vector or RXRα, 50 ng empty vector or CAR constructs and 10 ng pRL-CMV.
Figure 6.
Figure 6.
Transient transfection assays. (A) CAR AF-2 is required for transactivation through a DR-1 element. COS-1 cells were transfected with 100 ng p(rAox-PPRE)4-tk-Luc, 50 ng empty vector or RXRα, 50 ng empty vector or CAR constructs and 10 ng pRL-CMV. (B) CAR activation of p(rAox-PPRE)4-tk-Luc is RXRα DNA-binding domain dependent. COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(−) empty vector or pCDNA3.1(−)CAR plasmids, 50 ng pCDNA3.1(−), pCDNA3.1(+)RXRα or pCDNA3.1(+)RXRαΔDBD and 10 ng pRL-CMV (for transfection normalization) and assayed for luciferase activity 24–48 h post-transfection. (C) CAR transactivation of p(rAox-PPRE)4-tk-Luc is independent of RXRα AF-1 and RXRα AF-2. COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(−) empty vector or pCDNA3.1(−)CAR plasmids, 50 ng pCDNA3.1(−), pCDNA3.1(+)RXRα, pCDNA3.1(+)RXRαΔAF-1 or pCDNA3.1(+)RXRαΔAF-2 and 10 ng pRL-CMV (for transfection normalization) and assayed for luciferase activity 24–48 h post-transfection. (D) RXRα deletion constructs DBD- and AF-2-dependently transactivate p(rAox-PPRE)4-tk-Luc in response to RXR ligand 9-cis-retinoic acid (RA). COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(+) or pCDNA3.1(+)RXRα or RXRα domain deletion variants and 10 ng pRL-CMV (for transfection normalization) for 24 h, then treated with dimethyl sulfoxide (DMSO) solvent control or 1 μM 9-cis RA for 24 h and assayed for luciferase activity.
Figure 7.
Figure 7.
Transient transfection assays. (A) Interplay of RXRα homodimerization or heterodimerization with CAR in transactivation of p(rAox-PPRE)4-tk-Luc in the absence or presence of RXRα ligand 9-cis-RA. COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(−) or pCDNA3.1(−)CAR 1, 50 ng pCDNA3.1(+) or pCDNA3.1(+)RXRα or RXRα point mutants (Y397A heterodimerization mutant; L430F homodimerization mutant) and 10 ng pRL-CMV (for transfection normalization) for 24 h, then treated with dimethyl sulfoxide (DMSO) solvent control or 100 nM 9-cis-retinoic acid (RA) for 24 h and assayed for luciferase activity. (B) RXRα heterodimerization-dependent nuclear localization of CAR 1, 76, 80 in COS-1 cells. COS-1 cells were transfected with 200 ng pCDNA3.1(−) empty vector, CAR 1, 76 or 80 and 200 ng pCDNA3.1(−), RXRα, RXRαY397A (heterodimerization mutant) for 24–48 h. Immunofluorescent detection of CAR was performed as described in the Experimental procedures section.
Figure 8.
Figure 8.
Transient transfection assays. (A) 5α-Androstan-3α-ol (androstanol) dose–response to optimize downregulation of CAR 1-dependent transactivation of p(DR-4)3-tk-Luc. COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(−), pCDNA3.1(−)CAR 1 or CAR76, 50 ng pCDNA3.1(−)PPARα and pCDNA3.1(+)RXRα and 10 ng pRL-CMV (for transfection normalization) for 24 h. Cells were then treated with dimethyl sulfoxide (DMSO) solvent control or 0.5, 1, 2, 4 or 8 μM androstanol for 24 h and assayed for luciferase activity. (B) 5α-Androstan-3α-ol (androstanol) dose–response to optimize downregulation of CAR 1 and ΔNCAR-dependent transactivation of p(rAox-PPRE)4-tk-Luc. COS-1 cells were transiently transfected with 100 ng of reporter construct, 50 ng pCDNA3.1(−), pCDNA3.1(−)CAR 1 or CAR76, 50 ng pCDNA3.1(−)PPARα and pCDNA3.1(+)RXRα and 10 ng pRL-CMV (for transfection normalization) for 24 h. Cells were then treated with DMSO solvent control or 0.5, 1, 2, 4 or 8 μM androstanol for 24 h and assayed for luciferase activity. (C) CITCO dose–response to optimize re-activation of androstanol-antagonized CAR 1 and ΔNCAR activity on p(rAox-PPRE)4-tk-Luc. COS-1 cells were transiently transfected with 100 ng of reporter construct 50 ng pCDNA3.1(−), pCDNA3.1(−)CAR 1, CAR 76, CAR 80 or pCDNA3.1(−)PPARα and pCDNA3.1(+)RXRα and 10 ng pRL-CMV (for transfection normalization) for 24 h. Cells were then treated with DMSO solvent control, 2 μM androstanol, or co-treated with androstanol and increasing CITCO (0.3, 0.6, 1.25, 2.5 μM) for 24 h and assayed for luciferase activity.
Figure 9.
Figure 9.
Electrophoretic mobility shift assays. (A) CAR 1 and CAR 76 binding to [32P]NR1(DR-4) or [32P]rAox-PPRE. COS-1 cells were transiently transfected with pCDNA3.1(−), CAR 1 or CAR 76 and subjected to selection with 500 μg/ml G418 to enrich a population of cells overexpressing CAR 1 and CAR 76. Nuclear extracts were obtained from the CAR-expressing cells and used in EMSA as described in the Experimental procedures section. (B) CAR 1 and PPARα binding to [32P]NR1(DR-4) or [32P]rAox-PPRE. COS-1 cells were transiently transfected with pCDNA3.1(−) CAR 1 or pCDNA3.1(−)PPARα for 24 h, nuclear extracts were obtained and EMSA performed as described in the Experimental procedures section.
Figure 10.
Figure 10.
Lentiviral infections and real-time PCR analysis. (A) GFP expression in primary human hepatocytes infected with GFP-expressing lentivirus. Pseudoviral particles were produced using pSIH1-H1-copGFP lentiviral vector, virus was concentrated and infections were conducted as described in the Experimental procedures section. Images depict hepatocytes at 5 days post-infection in phase-contrast (100×), and fluorescence microscopy (100×). (B) Real-time PCR analysis of CAR, CYP2B6, PEPCK and HMGCS2 expression levels in primary human hepatocytes from two different donors infected with CAR1- and CAR76-expressing lentivirus. Pseudoviral particles expressing CAR1 and CAR76 in pCDH1-MCS1-EF1-copGFP lentiviral vectors were produced and infections were performed as described in the Experimental procedures section. Four days post-infection hepatocytes were harvested and RNA was isolated, converted to cDNA, subjected to real-time PCR and the data analyzed as described in the Experimental procedures section. Each bar depicts the average relative fold expression of two PCR reactions from a single hepatocyte donor.

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