Xenoreceptors of the nuclear receptor superfamily, such as pregnane X receptor (PXR), are liver-enriched ligand-activated transcription factors regarded as crucial sensors in xenobiotic exposure and detoxification. PXR controls transcription of many drug-handling genes and influx/efflux transporters, thus playing a crucial role in drug metabolism and excretion. Liver functions have been studied using primary human hepatocytes (PHHs), which, when conventionally cultured, undergo rapid de-differentiation, leaving them unsuitable for long-term studies. Recently, 3D PHHs called spheroids have emerged as an in vitro model that is similar to in vivo hepatocytes regarding phenotype and function and represents the first in vitro model to study the long-term regulation of drug-handling genes by PXR. In this study, we used mathematical modelling to analyze the long-term activation of PXR in 3D PHHs through expression kinetics of three key PXR-regulated drug-metabolizing enzymes, CYP3A4, CYP2C9, and CYP2B6 and the P-glycoprotein efflux transporter encoding gene, MDR1. PXR action in 3D PHHs was induced by the antibiotic rifampicin at two clinically relevant concentrations. The results confirmed that high rifampicin concentrations activated PXR nearly to its full capacity. The analysis indicated the highest PXR-induced transcription rate constant for CYP2B6. The rate constant dictating mRNA degradation associated with activated PXR was highest for CYP3A4. Moreover, we measured the metabolic activity of CYP3A4, CYP2C9, and CYP2B6 and quantified their metabolic rate constants. Metabolic activity rate constant of CYP3A4 was found to be the highest whereas that of CYP2B6 was found to be the lowest among the studied enzymes. Our results provide important insight into the regulation of PXR-target genes in 3D PHHs and show that mRNA expression and metabolic activity data can be combined with quantitative analysis to reveal the long-term action of PXR and its effects on drug-handling genes.

• MeSH
• biologické modely MeSH
• buněčné sféroidy * metabolismus   MeSH
• cytochrom P-450 CYP3A metabolismus   genetika   MeSH
• hepatocyty * metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• pregnanový X receptor * metabolismus   MeSH
• regulace genové exprese * MeSH
• rifampin farmakologie   MeSH
• steroidní receptory * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytochrom P-450 CYP3A MeSH
• pregnanový X receptor * MeSH
• rifampin MeSH
• steroidní receptory * MeSH

LIM and Src homology 3 (SH3) protein 2 (LASP2) is a small focal adhesion protein first identified as a splice variant of the nebulette gene (Nebl). As the newest member of the nebulin protein family, the regulation and function of LASP2 remain largely unknown. Our previous RNA-sequencing results identified Nebl as one of the most highly induced genes in the mouse liver in response to the activation of pregnane X receptor (PXR). In this study, we investigated this phenomenon further and show that PXR induces Lasp2 instead of Nebl, which partially use the same exons. Lasp2 was found to be induced in response to PXR ligand pregnenolone 16α-carbonitrile (PCN) treatment in mouse liver in vivo both after 4-day treatment and after long-term, 28-day treatment and in both male and female mice. Interestingly, the Lasp2 induction was more efficient in high-fat diet-fed mice (103-fold after 4-day PCN treatment) than in the normal chow-fed mice (32-fold after 4-day PCN treatment). Lasp2 induction was abolished in PXR knockout mice but could be rescued by re-expression of PXR, indicating that Lasp2 induction is PXR mediated. In mouse primary hepatocytes cycloheximide did not inhibit Lasp2 induction by PCN and a PXR binding site could be recognized upstream of the mouse Lasp2 gene suggesting direct regulation of Lasp2 by PXR. In human 3D hepatocytes, rifampicin induced only a modest increase in LASP2 expression. This study shows for the first time that PXR activation strongly induces Lasp2 expression in mouse liver and establishes Lasp2 as a novel PXR target gene. SIGNIFICANCE STATEMENT: RNA-sequencing results have previously identified nebulette (Nebl) to be efficiently induced by pregnane X receptor activating compounds. This study shows that instead of Nebl, LIM and Src homology 3 (SH3) protein 2 (Lasp2) coding for a small focal adhesion protein and partly sharing exons with the Nebl gene is a novel target of pregnane X receptor in mouse liver.

• Klíčová slova
• LASP2, Liver, Nebulette, Pregnane X receptor, Pregnenolone-16α-carbonitrile, Rifampicin,
• MeSH
• cytoskeletální proteiny * genetika   metabolismus   MeSH
• hepatocyty metabolismus   MeSH
• játra * metabolismus   účinky léků   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• pregnanový X receptor * metabolismus   genetika   MeSH
• pregnenolonkarbonitril farmakologie   MeSH
• proteiny s doménou LIM * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytoskeletální proteiny * MeSH
• pregnanový X receptor * MeSH
• pregnenolonkarbonitril MeSH
• proteiny s doménou LIM * MeSH

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related nuclear receptors with overlapping regulatory functions in xenobiotic clearance but distinct roles in endobiotic metabolism. Car activation has been demonstrated to ameliorate hypercholesterolemia by regulating cholesterol metabolism and bile acid elimination, whereas PXR activation is associated with hypercholesterolemia and liver steatosis. Here we show a human CAR agonist/PXR antagonist, MI-883, which effectively regulates genes related to xenobiotic metabolism and cholesterol/bile acid homeostasis by leveraging CAR and PXR interactions in gene regulation. Through comprehensive analyses utilizing lipidomics, bile acid metabolomics, and transcriptomics in humanized PXR-CAR-CYP3A4/3A7 mice fed high-fat and high-cholesterol diets, we demonstrate that MI-883 significantly reduces plasma cholesterol levels and enhances fecal bile acid excretion. This work paves the way for the development of ligands targeting multiple xenobiotic nuclear receptors. Such ligands hold the potential for precise modulation of liver metabolism, offering new therapeutic strategies for metabolic disorders.

• MeSH
• cholesterol metabolismus   krev   MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• dieta s vysokým obsahem tuků škodlivé účinky   MeSH
• hypercholesterolemie * farmakoterapie   metabolismus   etiologie   MeSH
• hypolipidemika * farmakologie   terapeutické užití   MeSH
• játra metabolismus   účinky léků   MeSH
• konstitutivní androstanový receptor MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pregnanový X receptor * antagonisté a inhibitory   metabolismus   genetika   MeSH
• receptory cytoplazmatické a nukleární * agonisté   metabolismus   genetika   antagonisté a inhibitory   MeSH
• žlučové kyseliny a soli metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cholesterol MeSH
• cytochrom P-450 CYP3A MeSH
• hypolipidemika * MeSH
• konstitutivní androstanový receptor MeSH
• pregnanový X receptor * MeSH
• receptory cytoplazmatické a nukleární * MeSH
• žlučové kyseliny a soli MeSH