Downregulation of HepaRG Cell CYP Genes by Hypoxia Inducible Factor Prolyl Hydroxylase (HIF-PH) Inhibitor

Abstract

Metabolic enzymes are occasionally downregulated in in vitro induction studies. Recently, HepaRG cells have been used for CYP induction assays instead of human hepatocytes in the early drug discovery stage; however, there is limited information on CYP downregulation by drug stimulation. In this study, we evaluated the effect of hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors, which downregulate CYP in human hepatocytes, on CYP gene expression in HepaRG cells. Microarray analysis to determine the expression levels of pharmacokinetics-related enzymes and RT-PCR to determine the expression levels of CYP3A4, CYP2B6, CYP1A2, and their nuclear receptor mRNA were conducted in HepaRG cells treated with HIF-PH inhibitors. Treatment of HepaRG cells with HIF-PH inhibitors decreased the expression of several pharmacokinetics-related metabolic enzymes, whereas Erythropoietin (EPO) and Pyruvate Dehydrogenase Kinase1 (PDK1) genes were induced. The expression of CYP3A4 and CYP2B6 in HepaRG cells showed concentration- and time-dependent downregulation following treatment with the HIF-PH inhibitor. The downregulation of these enzymes was correlated with the decrease of PXR/RXRα and CAR/RXRα, respectively. CYP1A2 decreased transiently, but recovered with continued HIF-PH inhibitor treatment. CYP3A4 and CYP2B6 were downregulated by HIF-PH inhibitors in HepaRG cells and human hepatocytes. In contrast, CYP1A2 in HepaRG cells responded differently to HIF-PH inhibitors than in human hepatocytes. Since CYP downregulation is commonly observed with HIF-PH inhibitors, along with the induction of EPO and PDK1 genes, stabilizing HIF may be one of the factors involved in CYP downregulation.

INTRODUCTION

In vitro CYP induction studies of the drug candidates were performed to predict potential drug interactions with concomitant drugs. Nuclear receptors such as Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are involved in CYP induction.¹⁾ These nuclear receptors recognize compounds and form dimers such as PXR/Retinoid X receptor (RXR), CAR/RXR, and AhR/ARNT, which are involved in the induction of CYP3A4, CYP2B6, and CYP1A2, respectively.

Hypoxia inducible factor (HIF) is increased in the case of hypoxia and is a transcriptional activator critical in local and systemic responses to hypoxia.²⁾ HIF has two subunits known as HIF-α and HIF-β. In the case of normoxia, HIF-α is first hydroxylated by HIF-prolyl hydroxylase (HIF-PH), and then undergoes ubiquitination and proteosome degradation. On the other hand, under hypoxic conditions, HIF-α is stabilized by the decreased activity of HIF-PH and is bound to HIF-β to form a functional dimer. It binds to hypoxic response elements in the nucleus and exhibits various physiological activities.^3,4)

Enarodustat is an oral HIF-PH inhibitor. It was developed and approved as a drug for improving renal anemia by inducing erythropoiesis and increasing and maintaining the hemoglobin concentration.⁵⁾ In our previous study, CYP3A4 and CYP1A2 mRNA expression were downregulated when Enarodustat was added to human primary hepatocytes and cultured.⁶⁾ According to the drug interaction guidance issued by the European Medicines Agency (EMA), U.S. Food and Drug Administration (FDA), and Pharmaceuticals and Medical Devices Agency (PMDA), CYP downregulation is described as follows: if concentration-dependent downregulation is observed in vitro and is not attributable to cytotoxicity, additional in vitro or in vivo studies may be recommended to understand the potential clinical consequences.^7–9) Due to many unclear aspects of the CYP downregulation mechanism, predicting how the compound will affect other pharmacokinetics-related enzymes is difficult.

HepaRG is a highly differentiated cell line with several human hepatocyte-like functions, expressing several CYPs, phase II enzymes, and transporter mRNAs.¹⁰⁾ In addition, it is known that HepaRG cells exhibit CYP3A4, CYP2B6, and CYP1A2 induction, as observed in human hepatocytes, by positive control compounds of enzyme induction such as Rifampicin and Omeprazol.^10,11) There are thus many reports of CYP3A4 induction studies in HepaRG cells^12–14) and these cells have been used as an alternative tool to human primary hepatocytes for evaluating the candidate compound’s ability to induce CYP3A4 during the early drug discovery stage. However, it is not clear whether HIF-PH inhibitors also cause the downregulation of CYP in HepaRG cells, and clarifying this point is important for understanding the mechanism of CYP downregulation by HIF-PH inhibitors in detail.

Since iron ions are required for hydroxylation of HIF-α by HIF-PH, cobalt chloride, which suppressed the utilization of iron ions, and deferoxamine, which forms complexes with iron ions, are frequently used in studies to clarify the involvement of HIF-PH.^2,15,16) When HepaRG or human fetal liver cells are cultured under hypoxic conditions in the presence of cobalt chloride and deferoxamine, it is reported that the mRNA expression of CYP is downregulated.^17,18) It has been also noted that the cytotoxicity to CYP by Cyclophosphamide and Ifosfamide are reduced in human medulloblastoma DAOY cells cultured under hypoxia conditions. This is because the expression levels of CYP2B6 and CYP3A4 were decreased in DAOY cells under hypoxic conditions, leading to the conversion of Cyclophosphamide and Ifosfamide to their cytotoxic metabolites.¹⁹⁾ It is important to know that CYP downregulation can be evaluated in cultured cells used in the early stages of drug discovery and in human hepatocytes by compound treatment.

In this study, we investigated the effects of HIF-PH inhibitor treatment on the pharmacokinetics-related enzymes in HepaRG cells by microarray analysis. In addition, the time- and concentration-dependent effects of HIF-PH inhibitors on CYP3A4, CYP2B6, and CYP1A2, which were downregulated in human hepatocytes, were investigated using HepaRG cells.

MATERIALS AND METHODS

Chemicals

Enarodustat was chemically synthesized by the Central Pharmaceutical Research Institute, Japan Tobacco (Osaka, Japan), and obtained from ChemScene (Monmouth Junction, NJ, U.S.A.). Roxadustat was obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.). Daprodustat was purchased from Selleck (Houston, TX, U.S.A.). Erythropoietin (EPO) was purchased from R&D Systems (Minneapolis, MN, U.S.A.). William’s Medium E and GlutaMAX were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.). HepaRG Thaw, plate, and general-purpose medium supplement (ADD670) and HepaRG induction medium supplement (ADD640) were obtained from Biopredic International (Saint-Grégoire, France).

Cell Culture

The HepaRG cells were purchased from Biopredic International. Cells were seeded at a density of 1.0 × 10⁵ cells/well in 96-well collagen type I coated plates using the HepaRG thawing medium, William’s Medium E with 2 mmol/L GlutaMAX I and supplement ADD670, and the cells were then incubated in a CO₂ incubator set at 37°C, 5% CO₂ for approximately 6 h. The culture medium was then replaced with fresh thawing medium, and HepaRG cells were incubated in a CO₂ incubator for 2 d. The culture medium was replaced with fresh HepaRG induction medium, William’s Medium E, containing 2 mM GlutaMAX I and supplemented with ADD640. Stimulation with the test compounds was initiated the following day.

Test Compound Treatments for Microarray Analysis

The test compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the plates in the cell culture medium at a final DMSO concentration of 0.1%. The HepaRG cells were treated for 24 h with Enarodustat, Roxadustat, and Daprodustat (10 μmol/L).

Microarray Analysis

HepaRG cell samples were lysed in QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) after 4 h in seeding plates and were then stored at −80°C until total RNA extraction. The HepaRG cell lysate was homogenized using a QIA shredder (QIAGEN). Total RNA was extracted and purified using the miRNeasy Micro Kit (QIAGEN). A Low Input QuickAmp Labeling Kit (Agilent Technologies, Santa Clara, CA, U.S.A.) was used for reverse transcription and preparation of labeled cDNA. Six hundred nanograms of cRNA was hybridized to SurePrint G3 Human GE microarrays (Agilent Technologies) for 17 h at 65°C. The arrays were washed and scanned using a SureScan microarray scanner (Agilent Technologies). Raw microarray data were processed and analyzed using a feature extraction system (Agilent Technologies). The gene expression levels in each sample were normalized across all samples using global normalization.

Cell Viability Assay

Viability of the cultured cells was determined using a Cell Counting kit-8 (DOJINDO, Kumamoto, Japan). After treatment with Enarodustat and Roxadustat at 10 and 30 μmol/L with HepaRG cells for 48 h, Cell Counting kit-8 was added to each well at a rate of 10% (v/v) and the plate was incubated in a CO₂ incubator for approximately 2 h. After incubation, the absorbance at 450 nm was measured using a GloMAX Discover Microplate Reader (Promega, Madison, WI, U.S.A.). The cell viability was calculated using the following equation:

% of cell viability = (A sample – A blank)/(A control – A blank) × 100

In this equation, A is the absorbance at 450 nm.

Test Compound Treatments for RT-PCR

The test compounds were dissolved in DMSO and added to the plates in the cell culture medium at a final DMSO concentration of 0.1%. The HepaRG cells were treated for 4, 8, 24, and 48 h with Enarodustat and Roxadustat (0.3–30 μmol/L). The medium, with or without test compounds, was replaced every 24 h.

EPO Treatments for RT-PCR

EPO was dissolved by adding phosphate-buffered saline (PBS) containing 0.1% serum albumin to prepare a stock solution at a concentration of 500 U/mL, which was then diluted 10000-fold to obtain the evaluation concentration. DMSO was added to the EPO treatment solution for cells at each concentration at a final concentration of 0.1%. The HepaRG cells were treated for 24 and 48 h with EPO (0.5–500 mU/mL). The medium, with or without test compounds, was replaced every 24 h.

Cell Lysate for RT-PCR

HepaRG cell lysates were prepared using a CellAmp Direct RNA Prep Kit for RT-PCR (TaKaRa Bio, Shiga, Japan).

Determination of mRNA Expression Levels

The mRNA levels of CYP1A2, CYP2B6, CYP3A4, AhR, ARNT, CAR, PXR, RXRα, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified using a quantitative RT-PCR. The primers were purchased from ThermoFisher Scientific (CYP1A2: Hs00167927_m1, CYP2B6: Hs03044633_m1, CYP3A4: Hs00604506_m1, AhR: Hs00169233_m1, ARNT: Hs01121918_m1, CAR: Hs00901571_m1, PXR: Hs011114267_m1, RXRα: Hs01067640_m1 and GAPDH: Hs99999905_m1). RT-PCR of HepaRG cells was performed using TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific), and mRNA levels were detected using StepOnePlus (Thermo Fisher Scientific). The mRNA expression levels were normalized to GAPDH gene expression, and the relative expression ratio was calculated by dividing the mRNA expression levels of the test compound by the mRNA expression levels of the vehicle control. In all the experiments, the mRNA expression level of GAPDH was not affected by the addition of endarodustat or roxadustat. When the relative expression ratio was 0.5 or less, it was judged that the mRNA expression level was presumably downregulated by the test compound.

Statistical Analysis

Statistical analysis was performed using Welch’s t-test or Dunnett’s multiple comparison test, depending on the experimental design, using EZR (Saitama Medical Centre, Jichi Medical University; https://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/satatmedEN.html; Kanda, 2012). All p-values were two-sided, and p-values of 0.05 or less were considered statistically significant.

RESULTS

Effect of HIF-PH Inhibitor on Pharmacokinetics-Related Genes Expression

Microarray analysis was performed for the HepaRG cells treated with Enarodustat, Roxadustat, and Daprodustat at 10 μmol/L for 24 h for investigating whether HIF-PH inhibitors affect pharmacokinetics-related enzymes and HIF-related genes. The metabolizing enzymes that were downregulated in response to all three compounds were Acyl-CoA oxidase (ACOX)2, CYP2B6, CYP2C19, CYP2C9, CYP2E1, CYP3A4, CYP7A1, Glutathione S-transferase (GST)A2, and Sulfotransferase (SULT)2A1 (Fig. 1). Among the genes whose expression was affected by HIF, heme, and iron transport, erythropoietin EPO and PDK1 mRNAs were induced by exposure to all three compounds (Fig. 2).

Fig. 1. Effect of HIF-PH Inhibitors on Pharmacokinetics-Related Enzymes Using Microarray Analysis

HepaRG cells were treated with Enarodustat, Roxadustat, or Daprodustat at 10 μmol/L for 24 h (n = 1 for each point); ACOX, Acyl-CoA oxidase; FMO, Flavin-containing monooxygenase; GST, Glutathione S-transferase; SULT, Sulfotransferase; UGT, Uridine diphosphate glucuronosyltransferase.

Fig. 2. Effect of HIF-PH Inhibitors on the Genes Whose Expression Is Affected by HIF, Heme, and Iron Transport Using Microarray Analysis

HepaRG cells were treated with Enarodustat, Roxadustat, or Daprodustat at 10 μmol/L for 24 h (n = 1 for each point); ALAS1, 5’-aminolevulinate synthase1; BACH, BTB and CNC homology; FEC, Ferrochelatase; HAMP, Hepcidin; HDAC, Histone deacetylase; HIGD, Hypoxia-induced gene domain protein; HO, Heme oxygenase; LONP, Lon protease; PDH, Pyruvate dehydrogenase; PDK, Pyruvate dehydrogenase kinase; SLC40A1, Ferroportin.

Cytotoxic Assessment

From the cell viability assay, HepaRG cells treated with Enarodustat and Roxadustat of 10 and 30 μmol/L for 48 h had similar cell viability as the control (Fig. 3). This result suggests neither compound showed cytotoxicity to HepaRG cells, even at 30 μmol/L for 48 h.

Fig. 3. Cell Viability in HepaRG Cells Treated with Enarodustat and Roxadustat

HepaRG cells were treated with Enarodustat or Roxadustat at 10 and 30 μmol/L and vehicle control (0.1% (v/v) DMSO) for 48 h. Viability of cultured cells was determined using the Cell Counting Kit-8. Data are shown as the mean ± standard deviation (n = 3 for each point).

Effect of HIF-PH Inhibitors on the Expression of CYP3A4, CYP1A2, and CYP2B6 mRNA

We investigated the effect of Enarodustat or Roxadusatat on the expression of CYP3A4, CYP1A2, and CYP2B6 mRNA of HepaRG cells treated with HIF-PH inhibitors at 10 μmol/L for 24 h. A decrease in the expression of CYP3A4 and CYP2B6 mRNA in a time-dependent manner was observed with both compounds (Figs. 4A, 4B, 4E, 4F). In contrast, CYP1A2 mRNA expression decreased after 4 and 8 h of short-term exposure to Enarodustat or Roxadustat, respectively. However, when the treatment was continued for 24 h or longer, CYP1A2 mRNA expression returned to the control levels (Figs. 4C, 4D).

Fig. 4. Effect of Enarodustat and Roxadustat at 10 μmol/L for Several Exposure Times on CYP3A4 (A, B), CYP1A2 (C, D), and CYP2B6 (E, F) mRNA Expression in HepaRG Cells

HepaRG cells were treated with Enarodustat or Roxadustat at 10 μmol/L for 4, 8, 24, and 48 h. Data are shown as the mean ± standard deviation (n = 3 for each point, n = 2 for the point of Roxadustat for 48 h concerning CYP2B6). Significant at *p < 0.05, **p < 0.01, vs. Control by Welch’s t-test.

When HepaRG cells were exposed to Enarodustat or Roxadustat at 0.3, 1, 3, 10, and 30 μmol/L for 24 h, the expression ratio of CYP3A4 and CYP2B6 mRNA was less than 0.5 at 10 μmol/L or more for Enarodustat and at 1 μmol/L or more for Roxadustat. The downregulation effects were concentration-dependent (Figs. 5A, 5C). The expression ratio of CYP1A2 mRNA was less than 0.5 at only 30 μmol/L for Roxadustat, whereas Enarodustat did not decrease CYP1A2 mRNA (Fig. 5B). The effects of compound treatment for 4, 8, 24, and 48 h at all tested concentrations on CYP3A4, CYP1A2, and CYP2B6 mRNA expression are shown in Supplementary Figs. S1–S3.

Fig. 5. Effect of Enarodustat and Roxadustat after 24 h Exposure at Several Concentrations on CYP3A4 (A), CYP1A2 (B), and CYP2B6 (C) mRNA Expression in HepaRG Cells

HepaRG cells were treated with Enarodustat or Roxadustat at 0,3, 1, 3,10, and 30 μmol/L for 24 h. Data are presented as the mean ± standard deviation (n = 3 for each point). Significant at *p < 0.05, **p < 0.01 vs. control by Dunnett’s test.

Comparison of Changes in CYP mRNA Expression and Nuclear Receptor Expression in HepaRG Cells Treated with HIF-PH

The relationships of changes in gene expression between PXR-CYP3A4, RXRa-CYP3A4, CAR-CYP2B6, RXRα-CYP2B6, AhR-CYP1A2, and ARNT-CYP1A2 in HepaRG cells treated with Enarodustat and Roxadustat were evaluated. The PXR-CYP3A4 and RXRα-CYP3A4 were positively correlated, with correlation coefficients of 0.662 and 0.806, respectively (Figs. 6A, 6B). Positive correlations were also observed between CAR-CYP2B6 and RXRα-CYP2B6, with correlation coefficients of 0.889 and 0.746, respectively (Figs. 6E, 6F). In contrast, the correlation coefficients of AhR-CYP1A2 and ARNT-CYP1A2 genetic fluctuations were −0.403 and −0.0711, respectively, and the expression changes in CYP1A2 did not correlate with those in AhR and ARNT (Figs. 6C, 6D).

Fig. 6. Relationships of Change in mRNA Expression between CYPs and Nuclear Receptors in HepaRG Cells

HepaRG cells were treated with Enarodustat or Roxadustat at 10 and 30 μmol/L for 4, 8, 24, and 48 h. The relationships between CYP3A4 and PXR (A), CYP3A4 and RXRα (B), CYP2B6 and CAR (C), CYP2B6 and RXRα (D), CYP1A2 and AhR (E), and CYP1A2 and ARNT (F) are shown. Differences in the colors of the symbols indicate differences in the compound exposure time. Yellow, light brown, dark brown, and red represent the samples treated for 4, 8, 24, and 48 h, respectively. The solid line represents the correlation at all time points. Data are shown as means (n = 3 for each point, n = 2 for the point of Enarodustat or Roxadustat for 48 h concerning CAR).

Effect of EPO on the Expression of CYP3A4, and CYP2B6 mRNA

We investigated the effect of EPO at 0.5, 5, 50, and 500 mU/mL for 24 and 48 h on CYP3A4 and CYP2B6 mRNA expression in HepaRG cells. Decreased expression of CYP3A4 mRNA was observed with EPO at 24 and 48 h (Fig. 7A). A tendency for EPO to decrease CYP2B6 mRNA was observed at concentrations from 5 to 500 mU/mL (Fig. 7B).

Fig. 7. Effect of EPO on CYP3A4 (A) and CYP2B6 (B) mRNA Expression in HepaRG Cells

HepaRG cells were treated with EPO at 0.5, 5, 50, and 500 mU/mL for 24 and 48 h. Data are shown as the mean ± standard deviation (n = 3 for each point). Dunnett’s multiple comparisons test was performed by comparing EPO treated cells with vehicle control cells in each treatment time (0.1% (v/v) DMSO) (**p < 0.01, ***p < 0.001).

DISCUSSION

HepaRG cells have been used as an alternative to human primary hepatocytes to evaluate the ability of candidate compounds to induce enzyme metabolism during early drug discovery. We have previously reported that Enarodustat showed a concentration-dependent downregulation of CYP3A4 and CYP1A2 mRNA expression in human hepatocytes.⁶⁾ It has also been reported that a series of HIF-PH inhibitors decreased the expression of CYP3A4, CYP1A2, and CYP2B6 mRNA in human hepatocytes, and their effects were related to a prolyl hydroxylase inhibition potential.²⁰⁾ On the other hand, in HepaRG cells, CYP3A4 mRNA expression has been reported to be downregulated after 24 h of exposure to deferoxamine or CoCl₂.¹⁸⁾ These results of deferoxamine and CoCl₂ in HepaRG cells indicated the involvement of iron transport and hypoxia for the decreased CYP3A4 mRNA; however, the similarity of the response to Enarodustat and Roxadustat between human hepatocytes and HepaRG was still unclear.

CYP3A is the most important enzyme involved in drug metabolism, but other CYP enzymes and conjugating enzymes have also been reported to contribute to drug elimination.^21,22) Therefore, information on various pharmacokinetics-related enzymes is necessary, depending on the concomitant drugs. We investigated the effects of HIF-PH inhibitor treatment on pharmacokinetics-related metabolic enzymes in HepaRG cells using microarray analysis. In this microarray analysis, we considered the genes that commonly varied across these three HIF-PH inhibitors to be the metabolic enzymes affected by HIF stabilization due to HIF-PH inhibition. However, since the analysis was conducted with n = 1, these data are exploratory and should be interpreted with caution. In this study, the metabolic enzymes whose gene expressions were decreased by HIF-PH inhibitors were ACOX2, CYP2B6, CYP2C19, CYP2C9, CYP2E1, CYP3A4, CYP7A1, GSTA2, and SULT2A1 (Fig. 1). It has been reported that the gene expression of CYP2C9, CYP2E1, CYP3A4, CYP7A1, GSTA2, and SULT2A1 decreased when HepaRG cells were cultured under hypoxic conditions for 24 h.¹⁸⁾ Considering the results of this study and previous studies conducted using HepaRG cells under hypoxic conditions, it is suggested that the decreased gene expression of the metabolic enzymes affected in this study is a response to the stabilization of HIF. In fact, the expressions of EPO and PDK1 in HepaRG cells were increased by treatment with HIF-PH inhibitors (Fig. 2). It has been reported that EPO mRNA in Hep3B cells, which are derived from human hepatoma, is induced by the stabilization of HIF following treatment with Enarodustat⁵⁾ and Roxadustat.²³⁾ Regarding PDK1, it has been reported that PDK1 protein expression is induced by the stabilization of HIF by culturing cells under hypoxic conditions.²⁴⁾ These results also support the involvement of HIF stabilization in the decreased expression of metabolic enzymes.

The CYP induction potential assessment of drug candidate compounds is generally carried out to examine the effect mainly on CYP3A4 involving PXR/RXR, CYP1A2 involving AhR/ARNT, and CYP2B6 involving CAR/RXR, based on the main induction mechanism.¹⁾ Therefore, we examined the effects of treatment time and concentration of Enarodustat or Roxadustat on CYP3A4, CYP1A2, and CYP2B6 gene expression in HepaRG cells. CYP3A4 and CYP2B6 mRNA expression showed a concentration- or exposure time-dependent downregulation upon treatment with Enarodustat or Roxadustat (Figs. 4A, 4B, 4E, 4F, 5A, 5C). These results suggest that HIF-PH inhibitors downregulate CYP3A4 and CYP2B6 in HepaRG cells, similar to human hepatocytes. In contrast, the expression of CYP1A2 mRNA was transiently decreased by both HIF-PH inhibitors (Figs. 4C, 4D). These results indicate that the response of HepaRG cells to CYP1A2 mRNA by HIF-PH inhibitors is different from that of other CYP species. Heintze et al. reported that when cytochrome p450 reductase was knocked out in HepaRG cells, the induction of CYP1A2 gene expression and protein levels was observed, which is not seen in human cell culture systems.²⁵⁾ Although the mechanism by which the knockout of CYP reductase leads to CYP1A2 induction is unclear, it is possible that the difference in responsiveness of CYP1A2 gene expression between HepaRG cells and human hepatocytes manifested as differences in response to HIF-PH inhibitors. Further investigation into the differences in responsiveness is necessary, but previous studies have not confirmed whether or not the recovery of CYP1A2 gene expression due to HIF-PH inhibitor treatment occurs in human hepatocytes. Therefore, the behavior of the CYP1A2 gene in HepaRG cells following treatment with HIF-PH inhibitors is considered to have very low extrapolative value to human clinical settings, and this is regarded as a limitation of using HepaRG cells.

To investigate the mechanism of CYP downregulation by HIF-PH inhibitors, we examined the relationship between changes in the expression of CYP3A4, CYP1A2, and CYP2B6 mRNA and their nuclear receptor mRNA after HIF-PH inhibitor treatment. CYP3A4 and its regulated nuclear receptors, PXR and RXRα, showed good correlation. Similarly, CYP2B6 and its regulated nuclear receptors, CAR and RXRα, showed good correlation. These results indicate that the downregulation of CYP3A4 and CYP2B6 by HIF-PH inhibitors may be caused by changes in PXR and RXRα, CAR and RXRα, respectively. Preiss et al. reported that knocking out both PXR and CAR in HepaRG cells results in decreased basal CYP3A4 gene expression levels.²⁶⁾ Additionally, Hariparsad et al. reported that knocking out PXR in human hepatocytes leads to decreased basal CYP3A4 gene expression levels.²⁷⁾ These reports suggest that the decreased gene expression of nuclear receptors involved in CYP expression may lead to decreased expression levels of the CYP gene. In the present study, the influence of nuclear receptors was only confirmed at the mRNA level, and neither protein level confirmation nor functional assays for nuclear receptors have been conducted, so it is necessary to clarify whether decreased nuclear receptor expression directly affects CYP expression or whether decreased CYP expression results from compensatory reactions to the decrease in nuclear receptor expression. In addition, it has been reported that the stabilization of HIF reduces the expression of PGC1α, a coactivator of PXR.²⁸⁾ Moreover, HIF has been reported to activate ERK in blood vessels and the liver.^29,30) The activation of ERK by epidermal growth factor (EGF) has been reported not only to suppress CYP3A4 induction via PXR but also to decrease CYP3A4 expression.³¹⁾ The downregulation of CYP from HIF stabilization may involve pathways mediated by nuclear receptors or signaling pathways like ERK, and further investigation is necessary to elucidate the mechanisms involved. In contrast, there was no correlation between changes in the expression of CYP1A2-AhR and CYP1A2-ARNT. It was reported that the expressions of PXR, RXRα, CAR, and ARNT mRNA, but not AhR mRNA, were decreased in human hepatocytes treated with HIF-PH inhibitors.²⁰⁾ In this study, the expression of PXR, RXRα, CAR, and ARNT mRNA was decreased in HepaRG cells stimulated by HIF-PH inhibitors, and these results were consistent with previous results in human hepatocytes, whereas the mRNA level of AhR was also decreased in HepaRG cells. The expression of AhR mRNA has been reported to decrease in HepaRG cells cultured under hypoxic conditions.¹⁸⁾ As a result, the response to AhR in HepaRG cells treated with HIF-PH inhibitors may differ from that in human hepatocytes. The reason for this is unclear, and further studies are required. Among the metabolizing enzymes whose decreased gene expression was detected by microarray analysis (Fig. 1), CYP2C9 and CYP2C19 are regulated by PXR and CAR, respectively. It is also known that SULT2A1 is regulated by PXR, GSTA2 is regulated by AhR, PXR, and Nrf2, and CYP7A1 is regulated by PXR, FXR, and SHP.^32,33) Additionally, the effects of HIF-PH inhibitor treatment on transporters in HepaRG cells are shown in Supplementary Fig. S4. Among the transporters whose gene expression was found to decrease following HIF-PH inhibitor treatment in HepaRG cells, BCRP and MDR1 are reported to be regulated by PXR, CAR, and AhR.³⁴⁾ Since the variations of nuclear receptors such as PXR, CAR, and AhR involved in the gene expression of metabolic enzymes, whose gene expression fluctuates following treatment with Enarodustat or Roxadustat, are confirmed, they could be affected by changes in nuclear receptors.

The decrease in CYP3A4 and CYP2B6 gene expression in HepaRG cells treated with HIF-PH inhibitors may be related to HIF stabilization and changes in the expression of nuclear receptors such as PXR and CAR. However, the process by which HIF stabilization leads to CYP downregulation and decreased expression of nuclear receptor genes has not yet been elucidated. As for EPO, which was confirmed to be induced by microarray analysis, multiple administrations of EPO to rats decreased the expression of Cyp3a2 mRNA.³⁵⁾ In contrast, there have been no reports on the effects of EPO addition on CYP gene expression in humans. Therefore, we treated HepaRG cells with EPO and examined its impact on CYP3A4 and CYP2B6 gene expression (Fig. 7). The expression levels of CYP3A4 and CYP2B6 mRNA decreased with EPO treatment, but this was not dependent on EPO concentration. Similar to the effects on Cyp3a2 mRNA expression in rats, EPO may also lead to decreased CYP gene expression; however, further investigation is necessary to clarify the details. The mechanisms of CYP downregulation, including HIF stabilization and the involvement of EPO, are currently under investigation.

CONCLUSION

Treatment of HepaRG cells with HIF-PH inhibitors decreased the expression of CYP3A4 and CYP2B6 mRNA, which have been reported to be downregulated in human hepatocytes, and also decreased the gene expression levels of drug-metabolizing enzymes such as ACOX2, CYP2C19, CYP2C9, CYP2E1, CYP7A1, GSTA2, and SULT2A1. In addition, it was found that the decline in CYP3A4 gene expression in HepaRG cells treated with the HIF-PH inhibitor may be due to a concurrent decrease in the nuclear receptor expression of PXR and RXRα, while the reduction in CYP2B6 expression may be associated with a simultaneous decline in CAR and RXRα expression. However, the response of CYP1A2 in HepaRG cells to HIF-PH inhibitor treatment differed from that of CYP1A2 in human hepatocytes. Therefore, the difference in the CYP1A2 response requires attention when evaluating its downregulation in HepaRG cells. Since the downregulation of CYP3A4 and CYP2B6 and the induction of EPO and PDK1, as confirmed in HepaRG cells, were commonly observed in HIF-PH inhibitors, it is considered that the stabilization of HIF causes CYP downregulation in HepaRG cells.

Acknowledgments

This research was not supported by any public, commercial, or not-for-profit agency grants.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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