Abstract
Solute carrier family 2 member 9 (SLC2A9) is a voltage-driven transporter that mediates cellular uptake and efflux of various substrates such as uric acid. Here, we investigate the role of E4 promoter-binding protein 4 (E4BP4), a transcription factor, in regulating hepatic SLC2A9 in mice. Effects of E4BP4 on hepatic SLC2A9 and other transporters were examined using E4bp4 knockout (E4bp4−/−) mice. Transporting activity of SLC2A9 was assessed using uric acid as a prototypical substrate. We found that three SLC genes (i.e., Slc2a9, Slc17a1, and Slc22a7) were upregulated in the liver in E4bp4−/− mice with Slc2a9 altered the most. E4bp4 ablation in mice dampened the daily rhythm in hepatic SLC2A9, in addition to increasing its expression. Furthermore, E4bp4−/− mice showed increased hepatic uric acid but reduced uric acid in the plasma and urine. Consistently, allantoin, a metabolite of uric acid generated in the liver, was increased in the liver of E4bp4−/− mice. E4bp4 ablation also protected mice from potassium oxonate-induced hyperuricemia. Moreover, negative effects of E4BP4 on SLC2A9 were validated in Hepa-1c1c7 and primary mouse hepatocytes. Additionally, according to luciferase reporter and chromatin immunoprecipitation assays, E4BP4 repressed Slc2a9 transcription and expression via direct binding to a D-box (-531 bp to -524 bp) in the P2 promoter. In conclusion, E4BP4 was identified as a novel regulator of SLC2A9 and uric acid homeostasis, which might facilitate new therapies for reducing uric acid in various conditions related to hyperuricemia.
SIGNIFICANCE STATEMENT Our findings identify E4BP4 as a novel regulator of SLC2A9 and uric acid homeostasis, which might facilitate new therapies for reducing uric acid in various conditions related to hyperuricemia.
Introduction
Solute carrier family 2 member 9 (SLC2A9, also known as GLUT9) is a voltage-driven transporter that mediates cellular uptake and efflux of various substrates such as uric acid, glucose, and fructose (Vitart et al., 2008; Wright et al., 2010; Lu et al., 2019). SLC2A9 is highly expressed in the liver and kidney, consistent with its role in elimination of endobiotics and xenobiotics (Lu et al., 2019; Ruiz et al., 2018). Two functional splice variants have been identified for SLC2A9, and they differ only in the length of amino terminus (Prestin et al., 2014; Augustin et al., 2004). Compared with the long form, the short form has a shorter amino terminus (19 vs. 34 amino acids) (Prestin et al., 2014; Augustin et al., 2004). Both forms can be found in the liver and kidney (Keembiyehetty et al., 2006). The long form (isoform 1) is distributed in the basolateral side and the short form (isoform 2) in the apical side of polarized renal epithelial cells (Kimura et al., 2014; Augustin et al., 2004), whereas both are distributed in the basolateral membrane of hepatocytes (Keembiyehetty et al., 2006). In addition to its role in transporting substances, SLC2A9 has been implicated in cancer development and Parkinson's disease (Gao et al., 2013; Han et al., 2019).
Uric acid is a main product of purine metabolism that mainly occurs in the liver. Because uric acid has antioxidant activity, high circulating levels are beneficial against oxidative stress (Sautin and Johnson, 2008). However, circulating uric acid level above the normal range (i.e., hyperuricemia) is frequently linked to poor health, including hypertension, gout, metabolic syndrome, nephropathy, and cardiovascular diseases (Sharaf El Din et al., 2017). The kidney plays a critical role in maintaining circulating uric acid concentration through both secretion and reabsorption by a network of uric acid transporters such as SLC2A9, human urate transporter 1 (URAT1), organic anion transporter 1 (OAT1), OAT3, OAT4, breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (Xu et al., 2017). In addition, the liver also has a role in uric acid homeostasis through hepatocyte uptake from the circulation that is mediated by SLC2A9 (Preitner et al., 2009; Lu et al., 2019). This is evidenced by the fact that liver-specific loss of Slc2a9 in mice causes severe hyperuricemia and hyperuricosuria (Preitner et al., 2009). Uric acid in the liver can be either excreted to the bile (by the exporters such as BCRP) for clearance or degraded to allantoin (a pathway varies from species to species) (Ristic et al., 2020).
E4BP4 (NFIL3) is a basic leucine zipper (bZIP) transcription factor. E4BP4 negatively regulates the transcription of target genes by competing for D-box binding with the proline-alanine rich (PAR) family of bZIP transcription factors and recruiting histone deacetylase 2 and histone methyltransferase G9a via a repression domain consisting of amino acids 299–363 (Zhao et al., 2021; Tong et al., 2013). The role of E4BP4 in the immune system has been recognized. For instance, it is a critical regulator of IgE class switching, interleukin-3 (IL-3)-mediated cell survival, natural killer and Th17 cell development (Motomura et al., 2011; Keniry et al., 2014). Additionally, E4BP4 is implicated in regulating circadian rhythms via repression of clock genes such as Periods (Ohno et al., 2007). Interestingly, E4BP4 integrates circadian clock and development of immune cells through inhibiting RORγ, a Th17-determining factor (Yu et al., 2013).
Pharmacokinetics plays a critical role in determining drug concentrations in the plasma and tissues, thereby profoundly affecting drug efficacy and side effects. Pharmacokinetic processes consist of absorption, distribution, metabolism, and excretion, which for most drugs depend on drug-metabolizing enzymes (DMEs) and drug transporters (Li et al., 2019). Studies in recent years have revealed E4BP4 as a key regulator of DMEs such as carboxylesterase 2 (CES2) and cytochrome P450 3A11 (CYP3A11) (Tong et al., 2019; Zhao et al., 2018). E4BP4 positively regulates CES2 enzymes through inhibiting their transcriptional repressor, REV-ERBα, whereas it represses Cyp3a11 transcription by binding to a D-box in gene promoter (Tong et al., 2019; Zhao et al., 2018). Therefore, it is of no surprise that E4BP4 is a determinant of metabolism and pharmacokinetics for some drugs, including irinotecan and midazolam (Tong et al., 2019; Zhao et al., 2018). However, whether E4BP4 regulates drug transporters and chemical disposition remains largely unknown.
In this study, we investigate the role of E4BP4 in regulation of hepatic SLC2A9 in mice. Effects of E4BP4 on hepatic transporters were examined using E4bp4 knockout (E4bp4−/−) mice. Transporting activity of SLC2A9 was evaluated using uric acid as a prototypical substrate. We demonstrated that E4BP4 inhibits hepatic SLC2A9 expression in mice through a direct transrepression mechanism.
Materials and Methods
Materials
Assay kit for uric acid was obtained from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Uric acid was purchased from Yuanye Biotech (Shanghai, China). ELISA kit for allantoin was obtained from Jianglai Biotech (Shanghai, China). Anti-SLC2A9, anti-OAT2 and anti-GAPDH antibodies were obtained from Proteintech (Chicago, IL). TRIzol reagent was purchased from Accurate Biotech (Hunan, China). E4bp4-containing pcDNA3.1 plasmid (or E4bp4 plasmid), siRNA targeting E4bp4 (siE4bp4) and a negative control, as well as Slc2a9-P1 (−2000/+100 bp) and Slc2a9-P2 (−2000/+100 bp) luciferase reporters (cloned into pGL4.10 vector) (sequences are shown in Table 1 and Supplemental Table 1) were obtained from TranSheep Biotech (Shanghai, China).
Mice
E4bp4-deficient mice were obtained from Dr. Masato Kubo (Motomura et al., 2011). Wild-type mice (C57BL/6) were obtained from HFK Biotech (Beijing, China). All mice were kept on a 12-hour light, 12-hour dark cycle (lights on at 6:00 AM or ZT0 and lights off at 6:00 PM or ZT12), and food and water were available ad libitum. ZT stands for zeitgeber time, which is conventionally used to describe the time in a 12-hour light/dark cycle in which lights are turned on at ZT0 and off at ZT12. Male mice (8-12 weeks old) were used for experimentation.
PCR Genotyping
PCR genotyping (primers shown in Table 1) was performed as described (Chen et al., 2021).
Hyperuricemia
E4bp4−/− and wild-type mice (n = 5 per group) were intragastrically treated with potassium oxonate (PO, 250 mg/kg) at ZT2 for 7 consecutive days. Urine samples were collected over a 0- to 24-hour period after last drug dosing. On Day 8, mice were sacrificed, and plasma samples and livers were collected. Uric acid and allantoin levels were determined with the commercial kits.
Isolation of Primary Mouse Hepatocytes
Mouse hepatocytes were isolated from wild-type and E4bp4−/− mice using a collagenase perfusion method (Zhang et al., 2018).
Cell Culture and Treatment
Hepa-1c1c7 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS and primary mouse hepatocytes in Dulbecco’s modified Eagle’s medium containing 10% FBS plus 1% penicillin/streptomycin. Cells were seeded in a six-well plate and grown to a confluence of about 70% and then treated with E4bp4 plasmid (2 μg) or siE4bp4 (50 nM) or control. After 1 or 2 days, cells were harvested for expression analyses.
Cellular Uptake Experiments
Cellular uptake of uric acid was carried out as described previously (Yu et al., 2018). Briefly, cells (Hepa-1c1c7 and primary mouse hepatocytes) with or without transfection were incubated with uric acid (25, 50, and/or 100 μM) in Hanks’ balanced salt solution buffer. At specific time points (15, 30, or 60 minutes), the buffer was aspirated. Cells were washed using ice-cold PBS and then solubilized in 50% methanol. After centrifugation, the supernatant was collected, and uric acid was assayed.
qPCR Assay
Total RNA was extracted with TRIzol reagent and used for cDNA synthesis. qPCR reactions and amplification were performed as previously described (Wang et al., 2020; Wang et al., 2018). Peptidylprolyl isomerase B (Ppib) was used as an internal control. Primers are listed in Table 1. Relative mRNA level was calculated using the 2−ΔΔCt approach.
Western Blotting and Luciferase Reporter Assay
Western blotting and luciferase reporter assay were performed as described (Chen et al., 2021). In Western blotting, protein expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as an internal control).
Chromatin Immunoprecipitation Assay
Chromatin Immunoprecipitation (ChIP) assays were performed as previously described (Wang et al., 2020). In brief, mouse livers were fixed in formaldehyde. After sample lysis, chromatin was sheared and immunoprecipitated using anti-E4BP4 or normal IgG. DNA fragments were collected and purified, followed by qPCR quantification with specific primers (Table 1).
Data Analyses
Data are shown as mean ± S.D. Statistical analyses were performed with Student’s t test (for two group comparisons) and with ANOVA (one way or two way) and Bonferroni post hoc test (for multiple group comparisons). P < 0.05 was considered as a statistically significant difference.
Results
Validation of E4bp4-Deficient Mice
Polymerase chain reaction genotyping of E4bp4 was performed with mouse tails using a specific primer set (Table 1). E4bp4−/− mice generated a 350-bp fragment, whereas the wild-type allele produced a 2.5-kb fragment (Fig. 1A). By using primers targeting the deleted sequence, we demonstrated with qPCR assays the loss of E4bp4 transcript (wild-type) in the livers of E4bp4−/− mice (Fig. 1B).
Altered SLC2A9 Expression and Uric Acid Levels in E4bp4-Deficient Mice
To assess potential effects of E4BP4 on SLC transporters, we determined hepatic expression of a number of SLC genes, many of which encode drug transporters, in E4bp4−/− mice. The sample-collecting time point was set at ZT2, when the E4BP4 protein is highly expressed (Narumi et al., 2016). According to qPCR assays, three SLC genes (i.e., Slc2a9, Slc17a1, and Slc22a7/Oat2) were upregulated in the liver in E4bp4−/− mice with Slc2a9 altered the most (Fig. 2). To test if alterations in Slc2a9 expression depend on the time of the day, we extended our examinations to six time points around the clock considering that E4bp4 is a circadian gene (Zhao et al., 2018). We found that Slc2a9 oscillated in a circadian time-dependent manner with lowest value at ZT18 in wild-type mice (Fig. 3A). E4bp4 ablation in mice increased the hepatic expression of Slc2a9 mRNA throughout the whole day and blunted its diurnal rhythm (Fig. 3A). Renal expression of Slc2a9 also varied according to the daily time but was unaffected in E4bp4−/− mice (Fig. 3B). In line with the changes in mRNA, SLC2A9 protein was elevated in the liver of E4bp4−/− mice, and its diurnal rhythm was dampened (Fig. 3C). By contrast, hepatic OAT2 protein was unaffected in E4bp4−/− mice (Fig. 3D) despite a slight change in the mRNA level (Fig. 2). Because SLC2A9 is a uric acid transporter (Lu et al., 2019), we examined whether uric acid homeostasis is disrupted in E4bp4−/− mice. E4bp4−/− mice showed reduced plasma and urine uric acid but increased hepatic uric acid (Figs. 4A and 4B). We also found that allantoin, a metabolite of uric acid generated in the liver, was increased in the liver of E4bp4−/− mice (Fig. 4B). These changes can be attributed to upregulation of hepatic SLC2A9 which mediates the uptake of uric acid to the liver from the blood circulation. Supporting the in vivo findings, E4bp4-deficicent hepatocytes showed increased SLC2A9 expression and enhanced uptake of uric acid according to in vitro uptake experiments (Fig. 5). Altogether, we identified E4BP4 as a key regulator of hepatic SLC2A9 and uric acid homeostasis.
E4bp4 Ablation Desensitizes Mice to Hyperuricemia
Because uric acid homeostasis was disrupted in E4bp4−/− mice, we further attempted to examine whether the mice have an altered susceptibility to hyperuricemia. To this end, we established hyperuricemia models by treating mice intragastrically with 250 mg/kg PO for 7 days. Administration of PO induced hyperuricemia in both E4bp4−/− and control mice. However, hyperuricemia was less severe in E4bp4−/− mice than in control mice, as evidenced by significantly lower plasma and urine uric acid in the knockout mice (Fig. 6A). The fold increase in circulating uric acid after PO treatment was greater in E4bp4−/− mice than wild-type controls (3.4- vs. 1.6-fold). Consistently, hepatic uric acid and allantoin were higher in E4bp4−/− mice than wild-type controls (Fig. 6B). Altogether, E4bp4 ablation protects mice from PO-induced hyperuricemia.
E4BP4 Negatively Regulates SLC2A9 Expression in Hepa-1c1c7 and Primary Mouse Hepatocytes
Next, regulation of SLC2A9 expression by E4BP4 was evaluated in both Hepa-1c1c7 (a mouse hepatoma cell line) cells and primary mouse hepatocytes by performing gain and loss-of-function experiments. It was validated that the overexpression plasmid can increase E4bp4 expression, and that the specific siRNA can reduce the expression of E4bp4 in Hepa-1c1c7 cells (Figs. 7A and 7D). Overexpression of E4BP4 in Hepa-1c1c7 cells significantly reduced the SLC2A9 mRNA and protein, and also reduced the cellular uptake of uric acid (Figs. 7A–7C). Conversely, E4bp4 knockdown enhanced the cellular expression of SLC2A9 and uptake of uric acid (Figs. 7C–7F). Similar effects of E4BP4 on SLC2A9 expression were observed in primary mouse hepatocytes (Figs. 8A–8F). Taken together, E4BP4 negatively regulates SLC2A9 in Hepa-1c1c7 cells and in primary mouse hepatocytes, congruent with the animal findings (Fig. 3).
E4BP4 Represses Slc2a9 Transcription
E4BP4 is a critical component of the auxiliary loop of molecular clock, functioning as a transcriptional repressor to inhibit the expression of clock-controlled genes through binding to D-box elements in their promoters (Zhao et al., 2021). Given that E4BP4 is a negative regulator of SLC2A9, we reasoned that E4BP4 may regulate SLC2A9 expression through a direct transcriptional mechanism. Of note, two major Slc2a9 transcripts with E4BP4 significantly enriched at their promoters were identified (Fig. 9A). These two isoforms are driven by their own promoters, named P1 and P2, respectively. Intriguingly, E4BP4 dose-dependently inhibited the activity of a Slc2a9-P2-driven luciferase reporter in Hepa-1c1c7 cells (Fig. 9B). In contrast, E4BP4 did not affect the activity of a luciferase reporter driven by Slc2a9-P1 (Fig. 9B). In silico analysis revealed a D-box element (−531/−524 bp) in Slc2a9-P2 (Fig. 9C), whereas, no D-box element was found in Slc2a9-P1. Also, Slc2a9 transcript driven only by P1 promoter remained unchanged in the liver of E4bp4−/− mice (Fig. 9D). Furthermore, the inhibitory effects of E4BP4 on the activity of Slc2a9-P2 luciferase reporter were abrogated when the D-box sequence was mutated (Fig. 9E). Moreover, ChIP assay demonstrated that E4BP4 protein was enriched at Slc2a9-P2 in wild-type liver; however, this enrichment was no longer available in the liver of E4bp4−/− mice (Fig. 9F). As expected, E4BP4 did not bind to Slc2a9-P1 or a nonspecific region in the wild-type and E4bp4-deficient livers (Fig. 9F). Taken together, E4BP4 negatively regulates Slc2a9 transcription via binding to a D-box (−531/−524 bp) within Slc2a9-P2 promoter.
Discussion
We have unraveled that multiple transporters, including Slc2a9, Slc17a1, and Slc22a7, are regulated by E4BP4 in mouse liver (Fig. 2). Importantly, we showed that increased expression of SLC2A9 led to enhanced hepatocyte uptake of its endogenous substrate uric acid, and thus to a lower level of circulating uric acid as well as attenuated chemical-induced hyperuricemia in E4bp4−/− mice (Figs. 4 and 6). Negative regulatory effects of E4BP4 on SLC2A9 were further validated in primary mouse hepatocytes and in Hepa-1c1c7 cells (Figs. 7 and 8). Based on combined assays of luciferase reporter and ChIP, E4BP4 represses Slc2a9 transcription via direct binding to a D-box (-531/-524 bp) in the P2 promoter (Fig. 9). Taken together, E4BP4 inhibits hepatic SLC2A9 expression and activity in mice through a direct transrepression mechanism, thereby impacting transporting and elimination of substrate molecules. Identification of E4BP4 as a novel modulator of SLC2A9 and uric acid homeostasis might facilitate new therapies for reducing uric acid in various conditions related to hyperuricemia.
It is a novel finding that Slc2a9 is cyclically expressed and its level oscillates according to time of day (Fig. 3A). Slc2a9 mRNA peaks at ZT6, whereas its protein peaks at ZT10 with a 4-hour phase delay (Fig. 3). This is normal, because a certain amount of time is required for the translation of mRNA to protein (Narumi et al., 2016). The circadian expression of Slc2a9 may be translated to circadian rhythm in liver uptake of uric acid, thereby contributing to daily oscillations in the circulating level of uric acid as noted in rodents and humans (Kanemitsu et al., 2017). The Slc2a9 rhythmicity was dampened in E4bp4-deficient mice, indicating E4BP4 as a circadian regulator of Slc2a9 (Fig. 3). However, E4bp4−/− mice retained a rhythmicity in SLC2A9 expression, suggesting that other factors play a role in regulating circadian expression of this transporter. Promoter analysis of the Slc2a9 gene using the JASPAR algorithm identified two potential E-box elements (located at -1221/-1216 bp and -716/-711 bp) on which the core circadian oscillators BMAL1 and CLOCK may act to generate oscillations in gene expression (Wang et al., 2019; Zhao et al., 2019). Thus, it is likely that BMAL1 and CLOCK are involved in regulating circadian expression of Slc2a9. However, this requires further validations.
Uric acid homeostasis is dependent on the rates of its production and elimination. Uric acid is mainly synthesized from purine by xanthine oxidase in the liver. The urate oxidase gene encodes uricase that metabolizes uric acid to allantoin in the liver. We found that hepatic xanthine oxidase and urate oxidase were unaffected in E4bp4−/− mice (Fig. 10A). Thus, the contribution of uric acid production to an altered plasma level of uric acid in E4bp4−/− mice can be excluded. Uric acid is primarily eliminated in the kidney (about two-thirds) through filtration, reabsorption, and secretion (Bobulescu and Moe, 2012). In addition, the liver is an organ where SLC2A9 is highly expressed and has a role in urate homeostasis through hepatocyte uptake from the circulation for hepatobiliary elimination (Lu et al., 2019). In the study of Preitner et al. (2009), liver-specific loss of Slc2a9 in mice causes severe hyperuricemia and hyperuricosuria. In line with this, circulating uric acid level was reduced in E4bp4−/− mice with an elevated expression of hepatic SLC2A9 (Fig. 3 & 4). Therefore, this study strongly supports the notion that uptake of uric acid by the liver is a contributing factor to uric acid homeostasis besides the disposal processes in the kidney.
We focused primarily on assessment of the effects of E4BP4 on SLC2A9 and uric acid disposition in the liver. This was because neither SLC2A9 nor other uric acid transporters (including URAT1/SLC22A12, SLC22A6/OAT1, SLC22A8/OAT3 and ABCG2/BCRP) were affected in the kidney of E4bp4−/− mice (Fig. 10B). The exact reason as to why E4BP4 has no effect on renal SLC2A9 remains unknown. However, transcriptional mechanisms for gene regulation are tissue dependent because the transcription factors and coregulators vary across tissues (Jones et al., 2013; Xu et al., 2009). Tissue-specific regulatory effects for E4BP4 were also noted in previous reports in which E4BP4 positively regulates Ces3 genes in the liver, whereas it may repress the expression of renal Ces3 (Zhao et al., 2018; Gachon et al., 2006). Combined in vivo and in vitro evidence has been provided here that E4BP4 regulates SLC2A9 in mouse liver to alter uric acid homeostasis. However, whether E4BP4 regulates SLC2A9 and uric acid disposition in humans requires further studies.
E4BP4 in general competes for binding to D-box with the PAR bZIP proteins to regulate gene transcription and expression. In general, the PAR bZIP proteins activate, whereas E4BP4 represses, gene transcription. Their opposing roles in gene regulation are also true for DMEs and transporters such as Fmo5, Mdr1a, and Mrp2, and are critical for maintaining circadian rhythmicity in target genes (Yu et al., 2019; Zhou et al., 2019; Chen et al., 2019). Thus, there is a possibility that PAR bZIP factors activate Slc2a9 transcription and contribute to its circadian rhythm in expression. E4BP4 plays a role in immune system and in inflammatory responses. In turn, E4BP4 expression can be modified by inflammatory factors including IL-3, -4, -6, and -15 (Kashiwada et al., 2010). Therefore, it is possible that E4BP4 expression is altered in immune and inflammatory diseases and uric acid homeostasis is disrupted in these diseases due to changes in SLC2A9 expression.
It is noteworthy that Slc2a9 gene has two major transcripts (splice variants), encoding two protein isoforms that differ in the length of amino terminus but do not differ in their functions (Keembiyehetty et al., 2006). Compared with the long form, the short form has a shorter amino terminus (19 vs. 34 amino acids) (Keembiyehetty et al., 2006). Because the qPCR primers for Slc2a9 analysis (Figs. 2, 3, 7, and 8) cannot distinguish the two transcripts, the reported level of Slc2a9 mRNA was the sum of both transcripts. Also, anti-SLC2A9 antibody used here reacts with both forms of SLC2A9 proteins, and thus the Western blot data measured the total level of SLC2A9 protein. We revealed that E4BP4 regulates transcription of Slc2a9 isoform 2 (P2, short form) via binding to a D-box within its promoter region (Fig. 9) but has no effect on Slc2a9 isoform 1 (P1, lacking in a D-box). Additionally, we showed that E4bp4 has no effect on Slc2a9 transcript driven by P1 promoter by designing a specific primer set. Therefore, the isoform 2 was responsible for upregulated Slc2a9 expression in E4bp4−/− mice.
In summary, our study reveals E4BP4 as a negative regulator of SLC2A9 in mice, affecting the uptake of uric acid by the liver and circulating uric acid level. Importantly, E4BP4 represses Slc2a9 transcription via direct binding to a D-box (-531/-524 bp) in the P2 promoter. Future investigations are suggested to study the role of human E4BP4 in regulating SLC2A9 and uric acid homeostasis.
Authorship Contributions
Participated in research design: Z. Wang, S. Wang, Wu.
Conducted experiments: Z. Wang, Gao, Ren.
Contributed new reagents or analytic tools: Z. Wang, Gao.
Performed data analysis: Z. Wang, Gao.
Wrote or contributed to the writing of the manuscript: Z. Wang, S. Wang, Wu.
Footnotes
- Received November 25, 2021.
- Accepted February 14, 2022.
This work was supported by National Natural Science Foundation of China [Grant 82003839], the Guangdong Basic and Applied Basic Research Foundation [Grants 2019A1515110892, 2020A1515010538, and 2021A1515011256], and the Project of Administration of Traditional Chinese Medicine of Guangdong Province of China [Grant 20212047].
↵1 Z.W. and L.G. contributed equally to this work.
↵This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- BCRP
- breast cancer resistance protein
- bZIP
- basic leucine zipper
- ChIP
- chromatin immunoprecipitation
- DME
- drug-metabolizing enzyme
- E4BP4
- E4 promoter-binding protein 4
- OAT
- organic anion transporter
- PAR
- proline-alanine rich
- SLC2A9
- solute carrier family 2 member 9
- Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics