ABSTRACT It was reported that antituberculosis medicines could induce liver damage via oxidative stress. In this study, we investigated the effects of rifampicin (RFP) on the membrane

expression of multidrug resistance-associated protein 2 (MRP2) and the relationship between oxidative stress and RFP-induced endocytosis of MRP2 in HepG2 cells. We found that RFP (12.5–50 

μM) dose-dependently decreased the expression and membrane localization of MRP2 in HepG2 cells without changing the messenger RNA level. RFP (50 μM) induced oxidative stress responses that

further activated the PKC-ERK/JNK/p38 (protein kinase C-extracellular signal-regulated kinase/c-JUN N-terminal kinase/p38) and PI3K (phosphoinositide 3-kinase) signaling pathways in HepG2

cells, and their expression was clearly affected by the changes in intracellular redox levels. Knockdown of clathrin or AP2 with small interfering RNA attenuated RFP-induced decreases of

membrane and total MRP2. We further demonstrated that RFP markedly increased the ubiquitin–proteasome degradation of MRP2 in HepG2 cells, which was mediated by the E3 ubiquitin ligase GP78,

but not HRD1 or TEB4. In conclusion, this study demonstrates that RFP-induced oxidative stress activates the PKC-ERK/JNK/p38 and PI3K signaling pathways that leads to clathrin-dependent

endocytosis and ubiquitination of MRP2 in HepG2 cells, which provides new insight into the mechanism of RFP-induced cholestasis. You have full access to this article via your institution.

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LYSOSOMAL MEMBRANE PERMEABILIZATION AS WELL AS CYTOPROTECTIVE AUTOPHAGY IN RESPONSE TO DNA-DAMAGING DRUGS Article Open access 29 December 2022 INTRODUCTION Oxidation and antioxidant systems

exist in a dynamic equilibrium under normal conditions in organisms. However, upon exposure to harmful factors or under pathological conditions, organisms produce excessive reactive oxygen

species (ROS) that overwhelm the elimination capacity of the antioxidant systems, which induces oxidative stress [1]. Oxidative stress can directly lead to tissue injury via lipid

peroxidation and can activate many cytokines through the protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) signaling pathway, which further promotes the development of tissue

damage. It has been discovered that antituberculosis drugs induce liver damage via oxidative stress associated with increases in cellular oxidants, such as the lipid peroxidation product

malondialdehyde (MDA), and with consumption of antioxidants, including superoxide dismutase (SOD) and glutathione (GSH) [1,2,3,4,5]. Rifampicin (RFP) is a first-line antituberculosis drug

recommended by the World Health Organization that is infamous for its hepatotoxicity, which is the main side effect of tuberculosis treatment and the most critical factor restricting the

clinical application of this drug [6, 7]. RFP causes liver damage mainly by cholestasis, with an obvious increase in serum bilirubin as the major clinical manifestation. This effect is

closely associated with hepatocyte canalicular transporters, such as multidrug resistance-associated protein 2 (MRP2), which localizes to the hepatocyte apical membrane [8, 9]. Previous

studies have shown that RFP, an activator of pregnane X receptor, promotes the excretion of bile salts by upregulating the expression of MRP2 in hepatocytes. However, this finding is

inconsistent with the clinical phenomenon of biliary siltation caused by RFP [10]. Interestingly, studies on rat models of cholestasis have shown that impaired MRP2-mediated transport

coincides with strongly decreased MRP2 protein levels and endocytic retrieval of MRP2 without any significant changes in _MRP2_ messenger RNA (mRNA) levels [11]. Disrupted canalicular

localization and decreased MRP2 protein expression without changes in _MRP2_ mRNA expression have also been observed in patients with chronic cholestatic disorder and hepatic failure,

suggesting that post-transcriptional regulation of MRP2, such as redistribution of MRP2 from the canalicular membrane into the cytosol (i.e., endocytic retrieval) followed by ubiquitination,

may be considered a decisive step contributing to decreased bile flow [12, 13]. Therefore, we speculated that RFP-induced cholestasis may be associated with endocytosis of MRP2 and that

this process is perhaps the critical reason for RFP-induced hepatotoxicity. However, there have been no relevant reports until now. Studies have shown that oxidative stress is sometimes

accompanied by cholestasis and may be a key factor for endocytosis of envelope proteins. Sekine and Horie [14] reported that MRP2 was internalized when acute oxidative stress arose in rat

livers and returned to the canalicular membrane after replenishment of intracellular GSH, which was related to the redox-sensitive balance of protein kinase A (PKA)/PKC activation. In the

present study, we studied the effects of RFP on MRP2 in HepG2 human hepatic epithelial cells and explored the underlying mechanisms, and we found that RFP initiated endocytosis and

ubiquitin–proteasome degradation of MRP2 on the hepatocyte membrane via oxidative stress. Our findings lay a foundation for further research on and prevention of RFP-induced cholestasis.

MATERIALS AND METHODS CELL CULTURE AND PROCESSING HepG2 cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were incubated in high-glucose Dulbecco’s modified

Eagle’s medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, CA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 ng/mL amphotericin B (Invitrogen, CA,

USA) in a humid atmosphere with 5% CO2 at 37 °C. The medium was changed every 24 h. Cells were seeded at a density of 2 × 105 cells per well into six-well plates 24 h before drug treatments.

Glutathione reduced ethyl ester (GSH-MEE), RFP, hydrogen peroxide (H2O2), MG132, probenecid, and dimethyl sulfoxide (DMSO) were all purchased from Sigma-Aldrich (St. Louis, MO, USA).

GSH-MEE (2 mM, dissolved in water) was added to the medium 1 h before RFP (12.5, 25, or 50 μM, dissolved in DMSO) was added. Probenecid (50 μM) was used as a positive control for MRP2

inhibition. An equal volume of DMSO was added as the RFP negative control, and 1 mM H2O2 was used as a positive control for oxidative stress. To test whether RFP can degrade MRP2 through the

ubiquitin–proteasome pathway, MG132 (5 μM) was added to the medium 12 h before harvesting the cells. The duration of RFP stimulation was 24 h. CELL TRANSFECTION Small interfering RNAs

(siRNAs) for clathrin, adaptor protein 2 (_AP2_), and _GP78_ and a negative control siRNA were purchased from Invitrogen. HepG2 cells at approximately 60% confluence were transiently

transfected with the indicated combinations of siRNAs using Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) in strict accordance with the manufacturer’s instructions. Then, 48 

h post transfection, Western blotting was used to detect the transfection efficiency [15]. MEASUREMENT OF OXIDATIVE STRESS INDICATORS The supernatant was collected after HepG2 cells were

disrupted by ultrasound for subsequent detection of GSH, SOD, and MDA (Solarbio, Beijing, China) levels with microassay kits as per the manufacturer’s directions. QUANTITATIVE REVERSE

TRANSCRIPTION-POLYMERASE CHAIN REACTION According to the manufacturer’s protocols, 1 µg of total RNA extracted from HepG2 cells with RNAiso Plus (TaKaRa, Dalian, China) was reverse

transcribed with a PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time quantitative PCR was performed with a SYBR® Premix Ex Taq™ II Kit (TaKaRa, Dalian, China)

in an Applied Biosystems 7500 Fast real-time PCR system to detect the mRNA levels of specific genes. Glyceraldehyde-3-phosphate dehydrogenase (_GAPDH_) was used as a reference gene to

normalize the data. The primers are described in Table 1. IMMUNOFLUORESCENCE ANALYSIS Immunofluorescence analysis was performed as previously described [16]. Briefly, after the samples were

fixed with 4% paraformaldehyde and blocked with phosphate-buffered saline containing 1% bovine serum albumin and 0.3% Triton X-100, the proteins were detected with primary antibodies

followed by goat anti-rabbit immunoglobulin G (IgG) Alexa® Fluor 488 (1:500 dilution, ab150077, Abcam) or goat anti-rabbit IgG Alexa® Fluor 568 (1:500 dilution, ab175471, Abcam) secondary

antibodies in the dark for 1 h. The primary antibodies included a rabbit MRP2 polyclonal antibody (1:100, sc-20766, Santa Cruz, CA, USA), a rabbit clathrin heavy-chain monoclonal antibody

(1:100, #4796, Cell Signaling), and a rabbit AP2α monoclonal antibody (1:100, #3215, Cell Signaling). The nuclei were stained with Hoechst 33342. Images were obtained with a confocal laser

scanning microscope (LSM510, Carl Zeiss, Jena, Germany) WESTERN BLOT ANALYSIS Total proteins or membrane proteins were extracted from HepG2 cells with high-efficiency RIPA tissue/cell lysis

buffer (R0010, Solarbio) or a MinuteTM Plasma Membrane Protein Isolation Kit (SM-005, Invent Biotechnologies), respectively. The samples (40 μg per well) were separated by 8% or 10% sodium

dodecyl sulfonate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% (w/v) nonfat milk, the blots were

probed with primary antibodies at 1:1000 dilutions. A rabbit MRP2 polyclonal antibody (sc-20766) was purchased from Santa Cruz, and a rabbit PKCα monoclonal antibody (ab32376), a PKCε

monoclonal antibody (ab124806), a PKCδ monoclonal antibody (ab182126), an GP78 antibody (ab227450), an anti-SYVN1/HRD1 antibody (ab170901), and an anti-MARCH6/TEB4 antibody (ab183533) were

purchased from Abcam. A phospho (p)-PI3K polyclonal antibody (#4228), a PI3K monoclonal antibody (#4249), a p-p44/42 MAPK (ERK1/2) monoclonal antibody (#4370), a p44/42 MAPK (ERK1/2)

monoclonal antibody (#4695), a p-SAPK/JNK antibody (#9251), a SAPK/JNK antibody (#9252), a p-P38 MAPK monoclonal antibody (#4511), a P38 MAPK antibody (#9212), a clathrin heavy-chain

monoclonal antibody (#4796), a p-AP2 monoclonal antibody (#7399), a GAPDH monoclonal antibody (#2118), a ubiquitin antibody (#3933) and a Na+/K+ ATPase antibody (#3010) were all obtained

from Cell Signaling. Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, ab6721, Abcam) was used as the secondary antibody. The signals were generated with an Enhanced

Chemiluminescence Detection Kit (Beyotime, Shanghai, China) and were detected with an automatic molecular imaging system (Tanon-5500). Densitometric analysis was performed with ImageJ

software. CO-IMMUNOPRECIPITATION ASSAY Co-immunoprecipitation (Co-IP) assays were conducted with a Pierce® Co-IP Kit (Thermo, 21649) according to the manufacturer’s specifications. Briefly,

HepG2 cells were lysed with lysis/wash buffer on ice. The supernatant was collected and quantified with a BCA Protein Assay Kit (PC0020, Solarbio). Then, 300 μg of lysate was added to a spin

column containing a resin slurry that had previously been combined with MRP2 antibody or IgG (as a negative control) and shaken overnight at 4 °C. After elution and centrifugation, the

proteins combined with the MRP2 antibody were obtained and analyzed by Western blotting. STATISTICAL ANALYSIS The data are presented as the mean±standard deviation (SD) from three

independent experiments. Statistical analysis was carried out with one-way analysis of variance with SPSS 12.0 software. A value of _P_ < 0.05 was considered to indicate statistical

significance. RESULTS EFFECTS OF RFP ON THE EXPRESSION OF MRP2 IN HEPG2 CELLS As shown in Fig. 1a, probenecid markedly decreased the _MRP2_ mRNA levels in the treated groups compared to the

0 μM group, while the _MRP2_ mRNA levels did not change significantly under RFP stimulation at doses of 12.5–50 μM. However, both probenecid and RFP stimulation reduced membrane MRP2

(MRP2-M) and total MRP2 (MRP2-A) expression (Fig. 1b). Similarly, immunofluorescence analysis revealed that the green fluorescence intensity indicating MRP2 expression on the cell membrane

was clearly downregulated under RFP and probenecid stress compared to control conditions (Fig. 1c). These results indicated that RFP regulated the expression of MRP2 at the

post-transcriptional level. RFP DECREASED THE MEMBRANE DISTRIBUTION OF MRP2 BY ACTIVATING OXIDATIVE STRESS IN HEPG2 CELLS Next, we detected oxidative stress markers, including GSH, MDA, and

SOD, in HepG2 cells treated with RFP at a dose of 50 μM. Compared to those in the control group, the concentrations of GSH (Fig. 2a) and SOD (Fig. 2b) were significantly decreased and those

of MDA (Fig. 2c) were markedly increased in the RFP and H2O2 groups. However, the addition of GSH-MEE reversed the changes in GSH, SOD, and MDA levels, indicating that RFP induced oxidative

stress in HepG2 cells. Similarly, HepG2 cells in the GSH-MEE pretreatment group showed stronger green fluorescence of MRP2 on the membrane than those in the RFP and H2O2 groups (Fig. 2d).

These results demonstrated that RFP decreased the membrane distribution of MRP2 via oxidative stress. RFP ACTIVATED THE PKC-ERK/JNK/P38 AND PI3K SIGNALING PATHWAYS VIA OXIDATIVE STRESS After

treatment with RFP and H2O2, the expression of PKCα, PKCδ, and PKCε (Fig. 3a, b) and the phosphorylation levels of JNK, P38, and ERK (Fig. 3a, c) were clearly upregulated, while p-PI3K was

significantly downregulated, in the treated groups compared to the control group. However, GSH-MEE successfully inhibited the decrease in p-PI3K and the activation of PKCα, PKCδ, PKCε,

p-JNK, p-P38, and p-ERK induced by RFP, indicating that RFP activated the PKC-ERK/JNK/p38 and PI3K signaling pathways via oxidative stress. RFP CAUSED CLATHRIN-DEPENDENT ENDOCYTOSIS OF MRP2

VIA OXIDATIVE STRESS Compared to the control treatment, treatment with RFP and H2O2 not only significantly increased the mRNA levels of clathrin and _AP2_ (Fig. 4a) but also upregulated the

protein expression of clathrin and p-AP2; however, these effects were suppressed effectively by GSH-MEE (Fig. 4b). Immunofluorescence analysis also revealed a higher abundance of clathrin

and AP2 in the RFP and H2O2 groups than in the control group of HepG2 cells. However, the fluorescent signals were clearly reduced by the addition of GSH-MEE (Fig. 4c). Co-IP experiments

showed that clathrin and AP2 were present in HepG2 cell lysates whether they were treated with RFP or not (input), while these proteins were absent in the IgG group. After capture with the

MRP2 antibody, clathrin and AP2 were detected under RFP stimulation but not under control conditions, suggesting that RFP promoted the interactions of MRP2, clathrin, and AP2 (Fig. 4d). To

further explore the effects of clathrin and AP2 on the expression of MRP2 under RFP stimulation, we carried out siRNA experiments. The expression of clathrin and AP2 was successfully

downregulated after transfection with si-clathrin and si-AP2, respectively (Fig. 5a–c). The MRP2-A and MRP2-M levels were significantly higher in both the clathrin siRNA + RFP group and the

AP2 siRNA + RFP group than in the siRNA NC + RFP group (Fig. 5a, d, e). Moreover, silencing clathrin and AP2 markedly inhibited the RFP-induced decrease in MRP2-M fluorescence, further

illustrating that the RFP-induced decrease in MRP2 was associated with clathrin-dependent endocytosis (Fig. 5f). TREATMENT OF HEPG2 CELLS WITH RFP CONTRIBUTED TO THE UBIQUITIN–PROTEASOME

DEGRADATION OF MRP2 As shown in Fig. 6a, ubiquitinated MRP2 was detected in immunoprecipitates with MRP2 antibody from HepG2 cells treated with RFP but not in those from control cells.

Compared with RFP stress alone, addition of the proteasome inhibitor MG132 along with RFP stress significantly increased the relative expression of MRP2. However, MG132 alone showed no

significant effect on MRP2 expression compared with the control treatment of DMSO alone (Fig. 6b). These results implied that the MRP2-M was degraded via ubiquitination after

clathrin-dependent endocytosis induced by RFP. Next, we found that RFP significantly upregulated the expression of GP78 but had little effect on the expression of HRD1 and TEB4 (Fig. 6c).

Furthermore, knocking out _GP78_ with siRNA clearly restrained the decrease in MRP2 induced by RFP, suggesting that GP78 was the key E3 ligase in the RFP-induced ubiquitination of MRP2-M

(Fig. 6d). DISCUSSION Antituberculosis drugs are the main cause of acute drug-induced liver injury [17]. The incidence of liver injury caused by RFP is approximately 2%, and the injury is

more severe when RFP is combined with isoniazid. Unfortunately, the mechanism has remained unclear until now. Several studies have demonstrated that abnormal expression, altered

localization, and dysfunction of hepatobiliary transporter proteins are important in the pathogenesis of intrahepatic cholestasis [18,19,20]. For example, mutations involving the ATP-binding

domains of MRP2 can lead to Dubin–Johnson syndrome, which is characterized by hyperbilirubinemia and elevated bile acid levels. In this study, we found that RFP decreased the membrane

localization of MRP2 via clathrin-dependent endocytosis and ubiquitin–proteasome degradation induced by oxidative stress in HepG2 cells. Our findings provide new ideas and methods for

preventing and reducing hepatotoxicity caused by RFP. MRP2 is encoded by _ABCC2_ and belongs to the superfamily of ATP-binding cassette transporter proteins [21]. Secretion of GSH and

anionic conjugates such as bilirubin and bile acid is the key feature of MRP2. Previous reports have shown that cholestasis induces upregulation of the expression of MRP2 to promote the

excretion of bile salts, which has been thought to be an adaptive response to liver damage caused by cholestasis. However, other researchers have reported that hepatic MRP2 expression in

cholestatic rodent models is clearly reduced without significant changes in mRNA expression [18, 22]. Similarly, human liver biopsies from inflammation-induced icteric cholestasis (mainly

cholestatic alcoholic hepatitis) have been found to display reduced MRP2 immunostaining despite the conservation of _MRP2_ mRNA levels [23]. In the current study, we found that RFP decreased

both MRP2-A and MRP2-M protein levels but did not modify _MRP2_ mRNA expression in HepG2 cells, indicating that post-transcriptional regulation most likely plays an important role in

regulating MRP2 functions (Fig. 1a–c). It has been demonstrated that oxidative stress is a key feature in most hepatopathies, which can result in cholestasis through actin cytoskeleton

disarrangement and further endocytic internalization of bile salt export pump (BSEP) and MRP2 [24]. Sekine and Horie [25] and Sekine et al. [26] reported that ethacrynic acid, which induces

acute oxidative stress in the rat liver, reduces GSH, elevates Ca2+, induces NO production, and activates novel PKC in a sequential manner, ultimately leading to MRP2 internalization; the

authors reported that these effects were reversibly regulated by the intracellular redox-sensitive balance of PKA/PKC activation. Chronic oxidative stress also decreases hepatic MRP2 protein

expression and disrupts the canalicular localization of MRP2 [12]. Here, we discovered that RFP induced oxidative stress in HepG2 cells, decreasing GSH and SOD levels and increasing MDA

levels. However, pretreatment of cells with GSH-MEE clearly reversed the changes in GSH, SOD, and MDA levels. Furthermore, replenishment with GSH-MEE successfully inhibited the decrease in

the green fluorescence of membrane-distributed MRP2 (Fig. 2a–d). These results clearly demonstrated that RFP decreased MRP2-M levels via oxidative stress in HepG2 cells. Researchers have

considered redox status imbalance to be a common trigger of the signaling pathway leading to MRP2 internalization [27]. Previous studies showed  that Ca2+-dependent PKC-MAPK pathways,

including the ERK, JNK, and P38-type signaling pathways, participate in hepatocanalicular dysfunction and cholestasis by promoting F-actin rearrangement and further endocytic internalization

of canalicular transporters in response to _tert_-butyl hydroperoxide-induced oxidative stress [28,29,30]. Moreover, estradiol-17β-_D_-glucuronide (E17G) activates the internalization and

sustained intracellular retention of BSEP and MRP2 through the conventional PKC/p38 MAPK and PI3K/ERK1/2 signaling pathways, respectively [31]. In the present study, we discovered that

RFP-induced oxidative stress was an upstream initiating factor triggering the PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells, which were inhibited significantly by pretreatment

with GSH-MEE (Fig. 3a–c). Under normal circumstances, the transport and recovery of membrane proteins are maintained in a dynamic state of equilibrium. Proteins are transported from Golgi

bodies to the cell surface, where they execute their biological functions, and are then returned to endosomes via endocytosis, in which clathrin-dependent endocytosis plays a critical role

[32]. The PKC-MAPK signaling pathway is necessary for activating clathrin-dependent endocytosis and determines the fates of membrane transporters. AP2, the link between clathrin and

transport proteins, is activated by P38 and can mediate the internalization and subsequent degradation of membrane transport proteins [33]. In E17G-treated isolated rat hepatocyte couplets,

significant increases in the colocalization of MRP2 with clathrin, AP2, and Rab5 have been reported [34]. Silencing of _AP2_ with siRNA in rat SCHs completely prevents E17G-induced

endocytosis of BSEP and MRP2. We found that the mRNA levels, protein expression, and fluorescence intensity of clathrin and AP2 were markedly upregulated in HepG2 cells treated with RFP and

H2O2, but downregulated in HepG2 cells pretreated with GSH-MEE before being treated with RFP (Fig. 4a–c). Co-IP experiments revealed that RFP increased the interaction of MRP2, clathrin, and

AP2 (Fig. 4d). However, knockdown of clathrin and _AP2_ with siRNAs successfully restrained the reductions in MRP2-M and MRP2-A (Fig. 5a–f). Taken together, the results suggest that

RFP-activated oxidative stress may disturb the balance between the transport and recovery of membrane proteins and trigger clathrin-dependent endocytosis via the PKC-ERK/JNK/p38 and PI3K

signaling pathways. Ubiquitination functions as an internalization signal that sends the modified substrate to endocytic/sorting compartments, after which the substrate is recycled to the

plasma membrane or degraded by a proteasome. Ubiquitination is a reversible post-translational modification involving conjugation of ubiquitin to targeted proteins through sequential

reactions mediated by E1, E2, and E3 ubiquitin ligases, which determines their intracellular fates [35]. Among the ubiquitin ligases, E3 ligases, including GP78, HRD1, and TEB4, play pivotal

roles in ubiquitination. HRD1 and TEB4 are involved in the degradation of the mutant BSEP in progressive familial intrahepatic cholestasis type II [36]. However, in patients with

obstructive cholestasis, GP78 rather than HRD1 and TEB4 participates in MRP2 internalization and degradation activated by liver PKCs, which further leads to Ezrin Thr567 phosphorylation

[37]. Aida et al. [13] explained that clathrin-mediated endocytosis of BSEP and the degradation of internalized MRP2 facilitated by ubiquitination are responsible for the disappearance of

cell surface-resident transporters. We thus investigated whether the RFP-induced decrease in MRP2 was related to ubiquitination. As shown in Fig. 6a–c, we discovered that RFP increased the

ubiquitin modification and proteasome degradation of MRP2, in which GP78, rather than HDR1 and TEB4, played an important role. Silencing of _GP78_ with siRNA significantly inhibited the

downregulation of MRP2 induced by RFP, which further demonstrated that GP78-mediated ubiquitination determined the fate of MRP2 under RFP stimulation (Fig. 6d). CONCLUSION Our current study

suggests that RFP-induced oxidative stress activates the PKC-ERK/JNK/p38 and PI3K signaling pathways, leading to clathrin-dependent endocytosis and ubiquitin–proteasome degradation of MRP2

in HepG2 cells. These findings provide new insights supporting exploration of the mechanisms of RFP-induced cholestasis and suggest that changing the peroxidation states in hepatocytes or

inhibiting the ubiquitination of MRP2 may prevent or alleviate the hepatotoxicity caused by RFP. This study was limited to investigating the effect of RFP on MRP2 at the cellular level in

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National Natural Science Foundation of China (Nos. 81470850 and 30900678) and the Key Project of Science and Technology of Chongqing (CSTC, 2009BB5159). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Infectious Disease, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China Bao-yan Xu * Institute of

Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China Xu-dong Tang, Jing Chen, Hong-bo Wu, Wen-sheng Chen & Lei Chen

Authors * Bao-yan Xu View author publications You can also search for this author inPubMed Google Scholar * Xu-dong Tang View author publications You can also search for this author

inPubMed Google Scholar * Jing Chen View author publications You can also search for this author inPubMed Google Scholar * Hong-bo Wu View author publications You can also search for this

author inPubMed Google Scholar * Wen-sheng Chen View author publications You can also search for this author inPubMed Google Scholar * Lei Chen View author publications You can also search

for this author inPubMed Google Scholar CONTRIBUTIONS All authors contributed to study design and to the analysis and interpretation of the data; BYX and XDT performed the experiments; BYX

wrote the paper; and LC  reviewed and edited the manuscript. All authors read and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Lei Chen. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Xu, By., Tang, Xd., Chen, J. _et al._

Rifampicin induces clathrin-dependent endocytosis and ubiquitin–proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells.

_Acta Pharmacol Sin_ 41, 56–64 (2020). https://doi.org/10.1038/s41401-019-0266-0 Download citation * Received: 18 October 2018 * Accepted: 30 May 2019 * Published: 17 July 2019 * Issue Date:

January 2020 * DOI: https://doi.org/10.1038/s41401-019-0266-0 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a

shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * rifampicin * cholestasis * MRP2 *

oxidative stress * PKC-ERK/JNK/p38 * PI3K * endocytosis * cholestasis * HepG2 cells