DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

Characterization of ligand-dependent activation of bovine and pig constitutive androstane (CAR) and pregnane X receptors (PXR) with interspecies comparisons.

1. Nuclear receptors CAR (NR1I3) and PXR (NR1I2) are major ligand-activated transcriptional regulators of xenobiotic metabolism and disposition and modulators of endobiotic metabolism. Differences in xenobiotic selectivity between the human and rodent receptors are well recognized but there is lack of such information on properties of CAR and PXR in important domestic animals. 2. The pig and bovine receptors were cloned and their ligand profiles were systematically compared to corresponding human and mouse forms utilizing a panel of xenobiotics and structural analysis. 3. Pig CAR and PXR resemble their human counterparts which can be rationalized by only modest amino acid changes between critical residues of the human ligand-binding pockets (H203Q for CAR, L210V and M243I for PXR). 4. In contrast, bovine CAR shows a blunted response to CAR agonists and inverse agonists. These changes are likely due to disruptive mutations at or near critical hydrogen bond-forming residues (N165I, Y326F). The unresponsiveness of bovine PXR to human- and mouse-selective agonists may be related to substitutions at important ligand-contacting residues R410Q and F305V, respectively. 5. Our findings have implications for regulation of drug-metabolizing enzymes and transporters and pharmacokinetics in cattle and pigs.

An update on the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR; NR1I3) has emerged as one of the main drug- and xenobiotic-sensitive transcriptional regulators. It has a major effect on the expression of several oxidative and conjugative enzymes and transporters, and hence, CAR can contribute to drug/drug interactions. Novel functions for CAR are also emerging: it is able to modulate the metabolic fate of glucose, lipids, and bile acids, and it is also involved in cell-cell communication, regulation of the cell cycle, and chemical carcinogenesis. Here, we will review the recent information available on CAR and its target gene expression, its interactions with partner proteins and mechanisms of action, interindividual and species variation, and current advances in CAR ligand selectivity and methods used in interrogation of its ligands.

Composition and microarchitecture of human trabecular bone change with age and differ between anatomical locations.

The microarchitecture of trabecular bone adapts to its mechanical loading environment according to Wolff's law and alters with age. Trabecular bone is a metabolically active tissue, thus, its molecular composition and microarchitecture may vary between anatomical locations as a result of the local mechanical loading environment. No comprehensive comparison of composition and microarchitecture of trabecular bone in different anatomical locations has been conducted. Therefore, the objective of this study was to compare the molecular composition and microarchitecture, evaluated with Fourier transform infrared (FTIR) microspectroscopy and micro-computed tomography (µCT), respectively, in the femoral neck, greater trochanter and calcaneus of human cadavers. Specimens were harvested from 20 male human cadavers (aged 17-82 years) with no known metabolic bone diseases. Significant differences were found in composition and microarchitecture of trabecular bone between the anatomical locations. Compositional differences were primarily observed between the calcaneus and the proximal femur sites. Mineralization was higher in the greater trochanter than in the calcaneus (+2%, p<0.05) and crystallinity was lowest in the calcaneus (-24%, p<0.05 as compared to the femoral neck). Variation in the composition of trabecular bone within different parts of the proximal femur was only minor. Collagen maturity was significantly lower in greater trochanter than in femoral neck (-8%, p<0.01) and calcaneus (-5%, p<0.05). The greater trochanter possessed a less dense trabecular bone microarchitecture compared to femoral neck or calcaneus. Age related changes were mainly found in the greater trochanter. Significant correlations were found between the composition and microarchitecture of trabecular bone in the greater trochanter and calcaneus, indicating that both composition and microarchitecture alter similarly. This study provides new information about composition and microarchitecture of trabecular bone in different anatomical locations and their alterations with age with respect to the anatomical loading environments.

NMR relaxation times of trabecular bone-reproducibility, relationships to tissue structure and effects of sample freezing.

Nuclear magnetic resonance (NMR) spectroscopy provides a potential tool for non-invasive evaluation of the trabecular bone structure. The objective of this study was to determine the reproducibility of the NMR relaxation parameters (T(2), Carr-Purcel-T(2), T(1ρ)) for fat and water and relate those to the structural parameters obtained by micro-computed tomography (µCT). Especially, we aimed to evaluate the effect of freezing on the relaxation parameters. For storing bone samples, freezing is the standard procedure during which the biochemical and cellular organization of the bone marrow may be affected. Bovine trabecular bone samples were stored at -20 °C for 7 days and measured by NMR spectroscopy before and after freezing. The reproducibility of NMR relaxation parameters, as expressed by the coefficient of variation, ranged from 3.1% to 27.9%. In fresh samples, some correlations between NMR and structural parameters (Tb.N, Tb.Sp) were significant (e.g. the relaxation rate for T(2) of fat versus Tb.Sp: r = -0.716, p < 0.01). Freezing did not significantly change the NMR relaxation times but the correlations between relaxation parameters and the µCT structural parameters were not statistically significant after freezing, suggesting some nonsystematic alterations of the marrow structure. Therefore, the use of frozen bone samples for NMR relaxation studies may provide inferior information about the trabecular bone structure.