Rapid maturation of the hepatic cell line Huh7 via CDK inhibition for PXR dependent CYP450 metabolism and induction

Bulutoglu, Beyza; Mert, Safak; Rey-Bedón, Camilo; Deng, S; Yarmush, Martin L.; Usta, O. Berk

doi:10.1038/s41598-019-52174-w

Cited by 8 publications

(7 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hepatoma-derived cell lines are commonly used to investigate NAFLD in vitro and include HepG2, Huh7, and HepaRG cells. However, these exhibit an abnormal genotype, lack population diversity, and demonstrate poor liver-specific metabolic competence causing these cell lines to be an insufficient choice in modeling CYP450 drug regulation [ 2 , 22 , 23 ]. In contrast, primary human hepatocytes (PHHs) exhibit high functionality relative to the human organ in vivo and are considered reliable for predictive toxicological and pharmacological results [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver

Rey-Bedón

Banik

Gökaltun

et al. 2022

Biomedicine & Pharmacotherapy

Self Cite

View full text Add to dashboard Cite

Drug-drug-interactions (DDIs) occur when a drug alters the metabolic rate, efficacy, and toxicity of concurrently used drugs. While almost 1 in 4 adults now use at least 3 concurrent prescription drugs in the United States, the Non-alcoholic fatty liver disease (NAFLD) prevalence has also risen over 25%. The effect of NALFD on DDIs is largely unknown. NAFLD is characterized by lipid vesicle accumulation in the liver, which can progress to severe steatohepatitis (NASH), fibrosis, cirrhosis, and hepatic carcinoma. The CYP450 enzyme family dysregulation in NAFLD, which might already alter the efficacy and toxicity of drugs, has been partially characterized. Nevertheless, the drug-induced dysregulation of CYP450 enzymes has not been studied in the fatty liver. These changes in enzymatic inducibility during NAFLD, when taking concurrent drugs, could cause unexpected fatalities through inadvertent DDIs. We have, thus, developed an in vitro model to investigate the CYP450 transcriptional regulation in NAFLD. Specifically, we cultured primary human hepatocytes in a medium containing free fatty acids, high glucose, and insulin for seven days. These cultures displayed intracellular macro-steatosis after 5 days and cytokine secretion resembling NAFLD patients. We further verified the model’s dysregulation in the transcription of key CYP450 enzymes. We then exposed the NAFLD model to the drug inducers rifampicin, Omeprazole, and Phenytoin as activators of transcription factors pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR), respectively. In the NAFLD model, Omeprazole maintained an expected induction of CYP1A1, however Phenytoin and Rifampicin showed elevated induction of CYP2B6 and CYP2C9 compared to healthy cultures. We, thus, conclude that the fatty liver could cause aggravated drug-drug interactions in NAFLD or NASH patients related to CYP2B6 and CYP2C9 enzymes.

show abstract

Section: Introductionmentioning

confidence: 99%

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver

Rey-Bedón

Banik

Gökaltun

et al. 2022

Biomedicine & Pharmacotherapy

Self Cite

View full text Add to dashboard Cite

show abstract

“…The hypothesis raised was that CDK2 phosphorylated Ser-350 to attenuate PXR to activate the CYP3A4 gene; therefore, CDK2 repression resulted in Ser-350 dephosphorylation and restored PXR's ability to activate the gene in confluent Huh-7 cells. A phosphorylation-mimicking PXR S350D mutant was unable to heterocomplex with RXRa and activate the CYP3A4 promoter (55,98). Consistent with this hypothesis, treatment with a CDK inhibitor up-regulated CYP3A4 expression (98).…”

Section: Lbd Phosphorylationmentioning

confidence: 59%

“…A phosphorylation-mimicking PXR S350D mutant was unable to heterocomplex with RXRa and activate the CYP3A4 promoter (55,98). Consistent with this hypothesis, treatment with a CDK inhibitor up-regulated CYP3A4 expression (98). Moreover, a phos-phorylation-mimicking single mutation of Ser-350 to Asp disabled PXR induction of drug-metabolizing enzymes in mouse livers (99).…”

Section: Lbd Phosphorylationmentioning

confidence: 70%

Nuclear receptor phosphorylation in xenobiotic signal transduction

Negishi

Kobayashi

Sakuma

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes (1,2). An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, while PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics which directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA binding domain (DBD). PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DBD phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand binding domain (LBD) to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

show abstract

“…Notably, while hPXR is DMD-MR-2022-000859-R1 one of only 5 human nuclear receptors (out of a total of 48) that does not preserve a Thr-38 like phosphorylation motif in the DBD, several phosphorylation residues including have been identified with many of them located within the LBD (Lin et al, 2008;Pondugula et al, 2009;Elias et al, 2014;Hu et al, 2020). Studies have shown that these residues could be phosphorylated by kinases such as cyclin-dependent kinase 2 (CDK2), PKA, PKC, and vaccinia related kinase 1 (VRK1); and phosphomimetic mutation of T57D and S350D markedly impaired hPXR-mediated CYP3A4 transactivation (Lin et al, 2008;Gotoh et al, 2017;Bulutoglu et al, 2019). Together, these results characterize PXR as another nuclear receptor that is regulated through both phosphorylation signaling and traditional ligand binding.…”

Section: Mechanisms Of Pb Activation Of Hpxrmentioning

confidence: 99%

Phenobarbital in Nuclear Receptor Activation: An Update

Men

Wang

2022

Drug Metab Dispos

View full text Add to dashboard Cite

Phenobarbital (PB) is a commonly prescribed anti-epileptic drug that can also benefit newborns from hyperbilirubinemia. Being the first drug demonstrating hepatic induction of cytochrome P450 (CYP), PB has since been broadly used as a model compound to study xenobioticinduced drug metabolism and clearance. Mechanistically, PB-mediated CYP induction is linked to a number of nuclear receptors such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and estrogen receptor α, with CAR being the predominant regulator. Unlike prototypical agonistic ligands, PB-mediated activation of CAR does not involve direct binding with the receptor. Instead, dephosphorylation of threonine 38 in the DNA-binding domain of CAR was delineated as a key signaling event underlying PB-mediated indirect activation of CAR.Further studies revealed that such phosphorylation sites appear to be highly conserved among most human nuclear receptors. Interestingly, while PB is a pan-CAR activator in both animals and humans, PB activates human but not mouse PXR. The species-specific role of PB in gene regulation is a key determinant of its implication in xenobiotic metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In this review, we summarize the recent progress in our understanding of PB-provoked transactivation of nuclear receptors with a focus on CAR and PXR. Significance statementExtensive studies using PB as a research tool have significantly advanced our understanding of the molecular basis underlying nuclear receptor-mediated drug metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In particular, CAR has been established as a cell signaling-regulated nuclear receptor in addition to ligand-dependent functionality. This mini-review highlights the mechanisms by which PB transactivates CAR and PXR.

show abstract

Rapid maturation of the hepatic cell line Huh7 via CDK inhibition for PXR dependent CYP450 metabolism and induction

Cited by 8 publications

References 39 publications

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver

Nuclear receptor phosphorylation in xenobiotic signal transduction

Phenobarbital in Nuclear Receptor Activation: An Update

Contact Info

Product

Resources

About