Browsing by Author "Kandel, Benjamin"

Now showing 1 - 1 of 1

Open Access
Comparative analysis of the nuclear receptors CAR, PXR and PPARα in the regulation of hepatic energy homeostasis and xenobiotic metabolism
(2014) Kandel, Benjamin; Zanger, Ulrich (Prof. Dr.)
Nuclear receptors (NRs), most notably the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR), regulate the transcription of several drug metabolizing enzymes and transporters (DMET) and thus represent important regulators of drug metabolism in the liver. Accordingly, the ligand dependent activation of these NRs by drugs and other xenobiotics contributes to the intra- and inter-individual variability of the drug detoxifying system. CAR and PXR were further shown to regulate the transcription of key enzymes involved in lipid and glucose metabolism. The NR peroxisome proliferator-activated receptor alpha (PPARa), a key regulator of fatty acid catabolism and target of lipid lowering fibrates, was recently identified as a direct regulator of cytochrome P450 3A4 (CYP3A4) and also potentially of other DMET genes. In this respect, CAR, PXR and PPARa are determinants of an overlapping number of liver functions including drug metabolism and energy homeostasis and are therefore associated with adverse drug reactions as well as liver disease like steatosis. Until now there have been no comparative studies investigating the transcriptomes of CAR, PXR and PPARa in humans. Therefore, a major focus of this study was to assess the genome-wide transcriptional changes provoked by these NRs in primary human hepatocytes (PHHs). To investigate human liver-specific gene expression and its regulation PHHs represent the most suitable available in vitro cell system. To identify the CAR-, PXR- and PPARa-specific genome-wide expression changes, hepatocyte cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARa (WY-14643) as well as DMSO (vehicle control). Afterwards, the mRNA expression in these samples was determined utilizing Affymetrix® microarrays. The obtained expression data were statistically evaluated to identify the genes that showed a differential expression in response to the agonist treatments and to investigate to which metabolic functions these genes contribute. The results of these experiments confirmed that CAR, PXR and PPARa regulated a highly overlapping but distinct set of genes coding for DMET. For example, according to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses expression of 10 DMET genes were shown to be regulated by all three NRs, whereas other DMET genes responded exclusively to the activation of one of the NRs. In addition several DMET related genes previously not shown to be regulated by CAR [like CYP2E1, sulfotransferase 1B1 (SULT1B1), UDP-glucuronosyltransferase 2B4 (UGT2B4) and cytochrome P450 reductase (POR)], PXR [like CYP2E1, alcohol dehydrogenases (ADHs), flavin containing monooxygenase 5 (FMO5) and glutathione peroxidase 2 (GPX2)] or PPARa like UBT2B4, ADH1s and FMO5) were identified to respond to the respective agonists. For PXR and CAR, this extends the list of genes by which these NRs influence drug metabolism and potentially contribute to drug-drug interactions (DDIs). The results obtained further specify the role of PPARa as a regulator of drug metabolism in vitro by increasing expression of, e.g., CYP3A4, 2B6, 2C8 and UGT1A1, thus pointing to a potential role of PPARa in adverse drug reactions in vivo. Furthermore, several genes coding for proteins involved in energy homeostasis, were identified as differentially expressed in response to PXR activation [e.g., pyruvate dehydrogenase kinase 4 (PDK4), glycogen synthase 2 (GYS2), carnitine palmitoyltransferase 2 (CPT2)], where such a relation was not reported so far. These results further expanded the knowledge of how PXR potentially impact fatty acid catabolism, gluconeogenesis and lipid de novo synthesis and provide interesting starting points to investigate how PXR activation contributes to altered glucose and lipid levels or disease like hepatic steatosis. Besides ligand-dependent regulation of nuclear receptors, post-translational modification has also been shown to influence the activity of liver-enriched NRs and expression of their target genes. In this context, protein kinase A (PKA) had been shown to repress CYP3A4 expression via PXR in a species-dependent manner, whereas the influence of PKA on the expression of other DMET genes had not been investigated in detail so far. The second part of this work therefore investigated the impact of PKA activation on the expression and activity of important drug metabolizing enzymes in a PXR- as well as a CAR-dependent manner. In this work PKA activation in primary human hepatocytes was identified as a determinant of drug metabolism in vitro by repressing PXR- and CAR-mediated or reducing basal expression and activity of CYP1A1, CYP2B6, CYP2C8 and CYP3A4, but also expression of ATP-binding cassette B1 (ABCB1) and UGT1A1. Using reporter gene assays, these observed effects could be linked to PKA-mediated repression of PXR and CAR activity that may involve phosphorylation of these NRs. It could be further shown that expression of DMET genes was also repressed by the fasting hormone glucagon, a physiologically relevant activator of PKA signaling, which was not investigated in humans so far. Due to the promiscuous ligand-specificity of PXR, which includes numerous compounds, drug treatment often leads to PXR activation, even with so-called “natural” compounds like St. John’s wort (SJW). It would thus be highly desirable to develop strategies in drug development to assess or circumvent the activation of NRs without compromising the pharmacological effects. Therefore, the last part of this work consists of an in vitro study to investigate synthetic acylated phloroglucinols, designed as substitutes for hyperforin, regarding their potential to activate PXR. Hyperforin the major active constituent of the plant SJW used to treat depressions was shown to exert its antidepressant properties via indirect inhibition of serotonin reuptake by selectively activating the canonical transient receptor potential channel 6 (TRPC6). In addition, hyperforin is associated with clinically relevant drug-drug interactions in patients that had taken SJW concomitantly with other drugs due to potent activation of the nuclear receptor PXR by hyperforin. The phloroglucinol derivatives investigated in this thesis had previously been evaluated for their bioactivity. It had been reported that five of the nine synthetic acylated phloroglucinols activate TRPC6 with similar potency as hyperforin. In this work, all these nine synthetic phloroglucinol derivatives were investigated in comparison to hyperforin and rifampicin for their potential to activate PXR. Hyperforin and rifampicin treatment of HepG2 cells co-transfected with a human PXR expression vector and a CYP3A4 promoter reporter construct resulted in potent PXR-dependent induction, while all TRPC6-activating compounds failed to show any PXR activation or to antagonize rifampicin-mediated CYP3A4 promoter induction. Hyperforin and rifampicin treatment of primary human hepatocytes resulted in highly correlated induction of PXR target genes, whereas treatment with the phloroglucinol derivatives elicited moderate gene expression changes that only weakly correlated to those of rifampicin treatment. The herein observed lack of PXR activation by the TRPC6 activating phloroglucinols was further supported by in silico pharmacophore modeling that did not indicate potent agonist or antagonist interactions for the TRPC6 activating derivatives and docking studies that suggested interaction of only one of these compounds. These in silico studies performed by Prof. Sean Ekins are published together with the results presented in this work (Kandel et al., 2014). This approach shows that strategies avoiding PXR activation are conceivable in drug development in order to prevent DDIs and improve drug safety. Taken together, these results further increase the number of genes by which CAR, PXR, and PPARa contribute to the regulation of drug metabolism and energy homeostasis. Moreover it was demonstrated that the PKA, which is involved in the transduction of the effects of, e.g., the hormone glucagon, represents a determinant of the drug detoxifying system in humans. Furthermore, a strategy could be presented, taking the example of the hyperforin derivates, which can be used to investigate and avoid DDIs in drug development. Such information will become imperative in future personalized medicine and the ever-present polypharmacy in order to handle intra- and inter-individual variability and to minimize drug failure or drug-drug interactions.