Characterization of porcine pregnane X receptor, farnesoid X receptor and their splice variants.
The pregnane X receptor (PXR; NR1I2) and the farnesoid X receptor (FXR; NR1H4) regulate the expression of many major metabolic enzymes. With the pig being used as a model for humans in metabolic and toxicological studies and also an important food animal, we characterized the transactivation profile of the porcine orthologs of these receptors, pgPXR and pgFXR. We compared the transactivation profiles of these receptors and their splice variants to their human orthologs using mostly endogenous ligands. Five alternatively spliced variants were identified for pgFXR as part of this study, while five alternatively spliced variants of pgPXR had been previously described. Insertions and deletions within these splice variants generated truncated proteins or proteins with altered tertiary structures, resulting in altered transactivation. Realtime polymerase chain reaction analyses showed that the pgPXR variants were present in liver cDNA samples from 3.33% to 7.92% of the total pgPXR, while the pgFXR variants were present from 1.92% to 9.26% of the total pgFXR. pgFXR was fairly evenly expressed in seven different tissues. In a luciferase reporter assay, wild-type pgPXR (pgPXR-WT) and human PXR (hPXR) responded to 12 common ligands, with similar levels of activation occurring for six of these. Wild-type pgFXR (pgFXR-WT) significantly responded to three ligands, two of which also activated hFXR. 3-Methylindole (skatole) was identified as a novel inverse agonist for pgPXR-WT and pgFXR-WT as well as porcine constitutive androstane receptor. None of the pgPXR splice variants (SVs) were active in the luciferase reporter assay on their own; pgFXR-SV1 was activated by chenodeoxycholic acid to a similar degree as pgFXR-WT. When co-transfected with their corresponding wild-type proteins, pgPXR-SV1 and pgFXR-SV1 significantly increased receptor transactivation. In conclusion, pgPXR-WT and pgFXR-WT both responded to ligands that activated their human orthologs, and some of the alternatively spliced variants significantly altered pgPXR and pgFXR transactivation at in vivo expression levels.
Gray
Matthew A
MA
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada.
Pollock
Callie B
CB
Schook
Lawrence B
LB
Squires
E James
EJ
D000818:Animals; D018076:DNA, Complementary / Q000302:isolation & purification; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D008099:Liver / Q000473:pathology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D000077297:Pregnane X Receptor; D012326:RNA Splicing; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000235:genetics*; D011987:Receptors, Steroid / Q000096:biosynthesis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D000099150:Receptor, Farnesoid X-Activated
D018076:DNA, Complementary; D008156:Luciferases; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D000099150:Receptor, Farnesoid X-Activated; C000625852:NR1I2 protein, human