ToxSci Advance Access originally published online on March 16, 2007
Toxicological Sciences 2007 97(2):407-416; doi:10.1093/toxsci/kfm054
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Involvement of Mitogen-Activated Protein Kinase Signaling Pathways in Microcystin-LR–Induced Apoptosis after its Selective Uptake Mediated by OATP1B1 and OATP1B3
* Department of Environmental Medicine
Department of Molecular Oncology
Department of Clinical Pharmacy and Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 890-8544 Kagoshima, Japan
Division of Tumor Biochemistry, German Cancer Research Center, 69120 Heidelberg, Germany
1 To whom correspondence should be addressed. Fax: +81-99-265-8434. E-mail: haruto{at}m3.kufm.kagoshima-u.ac.jp.
Received December 22, 2006; accepted March 9, 2007
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ABSTRACT |
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The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC50 6.6nM) and HEK293-OATP1B3 cells (IC50 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector–transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2µM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis.
Key Words: apoptosis; MAPK; microcystin-LR; OATP1B1; OATP1B3; PP2A.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microcystin-LR, a potent cyclic heptapeptide hepatotoxicin produced by several bloom-forming cyanobacteria, covalently bind the serine/threonine protein phosphatase (PP) 1 and 2A, thereby influencing regulation of balance between cellular protein phosphorylation and dephosphorylation (Dietrich and Hoeger, 2005
; Gehringer, 2004). The importance of the understanding about the toxicity of the microcystin-LR is increasing due to its potency of acute cytotoxicy and tumor-promoting activity restricted to the hepatocytes of animals and humans (Dietrich and Hoeger, 2005; Gehringer, 2004). It is thought that microcystin-LR affects hepatocellular viability through induction of changes in the cytoskeleton triggered by inhibition of the PPs (Eriksson, 1989) and partially through generation of reactive oxygen species (ROS) (Ding et al., 2001).
World Health Organization has set a provisional guideline value for microcystin-LR of 1.0 µg/l (in this case 1.0nM) in water supply (Gehringer, 2004). It has investigated the mechanism of cytotoxicity of the microcystin-LR by using primary cultured murine hepatocytes, alternatively using cultured human cell lines so far (Botha et al., 2004; Lankoff et al., 2003, in press; Zegura et al., in press; Zhan et al., 2004). It is known that microcystin-LR has poor membrane permeability except hepatocytes; therefore, more than 1000-fold higher concentrations compared with the guideline value have been used for in vitro study in cultured cell lines (Botha et al., 2004; Chong et al., 2000; Eriksson et al., 1990; Lankoff et al., 2003, in press; Zegura et al., in press; Zhan et al., 2004). ROS production may be dominant cause of apoptosis with microcystin-LR in the previous reports. (Ding et al., 2001).
At least three uptake transporters for organic anions have been identified at the sinusoidal membrane of human hepatocytes: organic anion transporting polypeptide (OATP)1B1 (Abe et al., 1999; Hsiang et al., 1999; König et al., 2000a), OATP1B3 (König et al., 2000b), and OATP2B1 (Tamai et al., 2000). Whereas OATP2B1 is expressed in a number of tissues, both OATP1B1 and OATP1B3 are expressed exclusively in hepatocytes and are thus important carriers of organic anions into human hepatocytes (Briz et al., 2003; Cui et al., 2001; Hagenbuch and Meier, 2003; König et al., 2000a,b).
OATP1B3, a protein of 702 amino acids, is structurally similar to OATP1B1, a protein of 691 amino acids, with an amino acid identity of 80%. In addition, OATP1B3 shares a number of transport substrates with OATP1B1, such as bromosulfophthalein (BSP), bile acids, sulfate and glucuronate conjugates, thyroid hormones, peptides, and drugs. It has been reported that the hepatocyte-specific OATPs can transport several peptides including cholecystokinin (CCK-8) (Ismair et al., 2001), [D-penicillamine2,5]-enkephalin (Nozawa et al., 2003), BQ-123 (Kullak-Ublick et al., 2001), and phalloidin (Fehrenbach et al., 2003; Meier-Abt et al., 2004). The ability of these OATPs to transport peptides makes them good candidate for the uptake of microcystin-LR into liver. Recently, both OATP1B1 and OATP1B3 as well as OATP1A2, which is predominantly expressed in endothelial cells of the blood-brain barrier, were identified as transporters for a derivative of microcystin, dihydromicrocystin-LR using the Xenopus oocyte transport system (Fischer et al., 2005). However, to date there is not any evidence to demonstrate that either OATP1B1 or OATP1B3 can function as microcystin-LR transporters in human hepatocytes and consequently induce cytotoxicity.
In humans, there are four major mitogen-activated protein kinases (MAPKs) that function as critical mediators of signal transduction pathways. They are comprised of a family of serine/threonine kinases, extracellular signal-regulated protein kinases (ERK) 1/2, c-Jun NH2-terminal kinases (JNK)/stress-activated protein kinases, p38 family kinases (p38), and ERK5. The ERKs are generally activated in responsive to mitogen or growth factor stimulation and contribute to proliferation, development, differentiation, and cell survival (Fang and Richardson, 2005). In contrast, both the JNK and the p38 are activated by a variety of cellular stress and cytokines, and activate downstream transcription factors, with the activation of JNK contributing to apoptosis, inflammation, and tumorigenesis (Nebreda and Porras, 2000; Tamura et al., 2002). The activation of p38 pathway mediates apoptosis, cell motility, chromatin remodeling, and osmoregulation (Bulavin et al., 1999; Nebreda and Porras, 2000). In addition to the direct activation of designated downstream kinases, there are considerable cross talk among the various MAPK signaling cascades with a three-tier network of the MAPK kinase kinase, MAPK kinase, and MAPK.
In this study, we therefore analyzed the ability of OATP1B1 and OATP1B3 to mediate microcystin-LR transport using HEK293 cells stably expressing OATP1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). We demonstrate that OATP1B1 or OATP1B3 is indispensable for the induction of the hepatocytotoxicity by microcystin-LR. Furthermore, we tried to elucidate the molecular basis and involvement of MAPK-mediated pathways for apoptosis by the microcystin-LR using HEK293-OATP1B3 cells.
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MATERIALS AND METHODS |
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Reagents.
Microcystin-LR was purchased from Alexis (Lausen, Switzerland). Fetal calf serum (FCS) was obtained from Cancera International (Canada). Minimum essential medium (MEM), phenol red–free MEM, protease inhibitor cocktail, and p-nitrophenyl phosphate (pNPP) solution were purchased from Sigma (St Louis, MO). U0126, SP600125, and SB203580 were purchased from Cell Signaling Technology (Beverly, MA), Promega (Madison, WI), and Sigma, respectively. Polyclonal rabbit antibodies against phospho-p44/42 MAPKs (ERK1/2) (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phosphorylation state–independent p44/42 MAPK (ERK1/2), SAPK/JNK, and p38 MAPK were purchased from Cell Signaling Technology. Monoclonal mouse antibody against
-tubulin was purchased from Santa Cruz (Santa Cruz, CA). Polyclonal rabbit antibodies ESL against human OATP1B1 and SKT against OATP1B3 were arisen as earlier (König et al., 2000a,b). Secondary anitibodies against mouse IgG and rabbit IgG were purchased from Amersham (Buckingamshire, United Kingdom). Microcystin ELISA kits and sodium butyrate were purchased from Wako (Osaka, Japan). Bicinchoninic acid assay kit were purchase from Pierce (Rockford, IL).
Cell culture.
Human embryonic kidney cells, HEK293 cells, were permanently transfected with SLCO1B1 (HEK293-OATP1B1 cells) or SLCO1B3 (HEK293-OATP1B3 cells), which were generated earlier (König et al., 2000a; Letschert et al., 2004) and cultured in MEM supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin (MEM-10% FCS), and 400 µg/ml G418 at 37°C, 95% humidity, and 5% CO2.
Uptake study of microcystin-LR.
Cells were seeded in six-well plates precoated with 0.1 mg/ml poly-D-lysine at a density of 2 x 106 cells/well and cultured with 10mM sodium butyrate, which is a histone deacetylases inhibitor for 24 h. Before the uptake experiments, cells were washed with prewarmed (37°C) uptake buffer (12.5mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.3, 142mM NaCl, 5mM KCl, 1mM KH2PO4, 1.2mM MgSO4, 1.5mM CaCl2, 5mM glucose, 12.5mM KH2PO4). Transport studies were started by the addition of several concentrations of microcystin-LR in 1 ml of prewarmed uptake buffer to the cells. After aspiration the microcystin-LR–containing uptake buffer, transport of microcystin-LR was stopped by the addition of 1 ml of ice-cold 0.5% bovine serum albumin (BSA) in uptake buffer. Cells were washed twice with ice-cold 0.5 % BSA in uptake buffer and subsequently washed three times with 1 ml of ice-cold uptake buffer and further washed with 1 ml of ice-cold phosphate-buffered saline (PBS). Cells were harvested into 1 ml of ice-cold PBS. The harvested cells were centrifuged at 1500 rpm at 4°C for 3 min. The cells were added 250 µl of hypotonic lysis buffer (10mM Tris-Cl, pH 8.0, 10mM KCl, 1.5mM MgCl2), and left for 30 min at room temperature. The cells were homogenized and heated for 10 min at 95°C to denature the intracellular microcystin-binding protein and centrifuged at 13,000 rpm for 15 min at room temperature. The supernatants (25 µl) were applied to ELISA analysis for microcystin-LR. ELISA analysis for microcystin-LR was performed according to the manufacturer's instructions, and microcystin-LR determinations were compared with the supplied standard calibrators. Protein concentrations were determined by the BCA assay kit using 20 µl of the supernatant. For inhibition studies, transport was started by the addition of 1 ml of prewarmed 1µM microcystin-LR with inhibitors and performed assay as well as normal uptake study.
Accumulation study of microcystin-LR.
Cells (5 x 106) were seeded into six-well plates precoated with 0.1 mg/ml of poly-D-lysine and cultured for 2 days in the CO2 incubator. Cells were further incubated with several concentrations of microcystin-LR in 1 ml of MEM-10% FCS for 2 h at 37°C in the CO2 incubator. After incubation, the cells were washed three times with ice-cold PBS and then harvested into ice-cold 1 ml PBS. The harvested cells were centrifuged at 1500 rpm at 4°C for 3 min. The cells were added 300 µl of hypotonic lysis buffer and left for 30 min at room temperature. The cells were homogenized and heated for 10 min at 95°C and centrifuged at 13,000 rpm for 15 min at room temperature. The supernatants (25 µl) were applied to ELISA analysis for microcystin-LR.
PP assay.
Cells (5 x 106) were seeded into six-well plates precoated with 0.1 mg/ml of poly-D-lysine and cultured in phenol red–free MEM-10% FCS for 2 days in the CO2 incubator. Cells were further incubated with several concentrations of microcystin-LR for 6 h in the CO2 incubator. The cells were washed twice with ice-cold 0.9% NaCl and washed once with ice-cold buffer A (50mM Tris-Cl, pH 8.3, 15mM MgCl2, 5mM EDTA, and 5mM dithiothreitol). After wash, the cells were harvested and suspended in 500 µl of ice-cold buffer A containing 1% (vol/vol) protease inhibitor cocktail, 1% (vol/vol) NP-40, and 1mM Na3VO4. After centrifugation at 13,000 rpm for 15 min at 4°C, cell lysates were obtained. Two hundred microliters of the cell lysate was added and mixed with 50 µl of pNPP solution and then incubated for 60 min at 37°C. After incubation, absorbance at 405 nm was read.
Measurement of apoptotic cells.
Apoptotic cells were quantitatively evaluated by flow cytometry. For Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis, cells were cultured for 24 h in the presence of microcystin-LR and then trypsinized. The cells were centrifuged at 1500 rpm for 3 min at 4°C after dilution with MEM-10% FCS and further washed once with PBS. After wash, the cells (1 x 106) were applied to FACScan analysis using Annexin V-FITC Apoptosis Detection Kit (BioVision, CA) according to the instruction of the kit.
For the analysis of sub-G1 proportion, cells were prepared identically to that one for Annexin V-FITC/PI analysis and were suspended in 100 µl of PBS, thoroughly mixed with 100 µl of Coulter DNA-Prep LPR (Coulter, Miami, FA), and then 2 ml of Coulter DNA-Prep Stain was added and again mixed thoroughly. The mixtures were incubated for 15 min at room temperature. The DNA content and the sub-G1 fraction, representing apoptotic cells, were determined as previously described (Kitazono et. al., 1998).
The levels of fluorescent staining of the cells were analyzed with a Beckman EPICS Flow Cytometry (Coulter Electronics, Hialeah, FL).
Immunoblot analysis.
Crude membrane fractions were prepared from cultured transfected cells and were analyzed by immunoblotting as described earlier (Komatsu et al., 2000). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions. Samples were incubated for 5 min at 95°C, alternatively in the case of OATP1B1 and OATP1B3, for 30 min at 37°C to reduce aggregation of OATP1B1 and OATP1B3, prior to electrophoresis. Transfer to polyvinylidene difluoride (PVDF) membranes was performed electrophoretically for 30 min at 15 V (constant voltage) using a semidry blotting system (Transblot SD apparatus, Bio-Rad, Richmond, CA). The membrane was blocked with 5% BSA in TTBS (10mM Tris-Cl, pH 8.0, 0.35M NaCl, 0.05% Tween 20) for 1 h at room temperature and then incubated overnight at 4°C with antibodies against human OATP1B1, OATP1B3, ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, p38, phospho-p38, and -tubulin. The membrane was washed three times with TTBS and then incubated for 60 min with horseradish peroxidase–conjugated secondary antibodies for detection of interests. In the case of phosphoproteins, Can Get Signal for IB (Toyobo, Osaka, Japan) was used for the detection. The PVDF membrane was rinsed twice and then washed four times for 5 min with TTBS and then evenly covered with the ECL Western blotting detection reagents (Amersham) for 1 min. The membrane was immediately exposed to x-ray film (Hyperfilm ECL, Amersham) in a film cassette at room temperature for various periods.
Cytotoxicity studies.
Colorimetric assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to assess the sensitivity of the cells to microcystin-LR in vitro as described (Komatsu et al., 2000). Exponentially growing cells were trypsinized and harvested, and equal numbers (1.6 x 104) of cells in 180 µl of MEM-10% FCS were inoculated into each well of a 96-well microplate. After incubation overnight, 20 µl of microcystin-LR solutions were added to the cultures, and then they were incubated for 3 days. Thereafter, 50 µl of 1 mg/ml MTT solution was added to each well, and the plates were incubated for 3 h at 37°C in a CO2 incubator. After aspirating the culture medium, the resulting formazan was dissolved with dimethylsulfoxide. Plates were placed on a shaker for 5 min and read immediately at 570 nm with a microplate reader, MPR-A4i (Tosoh, Tokyo, Japan), and cell viability was determined. The IC50 value was determined as the concentration of microcystin-LR that reduced the viability of the cells to 50% of that in control medium.
For inhibition studies, cells in 170 µl of MEM-10% FCS were pretreated with 10 µl of OATP1B3 inhibitors using the case of BSP, rifampicin, and CsA, for 2 min, alternatively of other reagents such as U0126, SP600125, and SB203580, for 1 h, and then incubated for 3 days with 20 µl of microcystin-LR solution. In the case of N-acetyl-L-cysteine (NAC), cells were pretreated with 20 µl of microcystin-LR for 1 h, then supplementarily added 10 µl of NAC, and then incubated for 3 days. The dose-modifying factor (DMF) was calculated as follows: the IC50 value of microcystin-LR for cells was divided by the IC50 value of microcystin-LR combined with the inhibitors.
Statistical analysis.
Differences between groups were analyzed by Student's t-test. p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of OATP1B1 or OATP1B3
The OATP1B1 or OATP1B3 protein were detected in the HEK293 cells stably transfected with SLCO1B1 cDNA (HEK293-OATP1B1 cells) or SLCO1B3 cDNA (HEK293-OATP1B3 cells) by the immunoblot analysis (Fig. 1). In contrast, neither OATP1B1 nor OATP1B3 protein was detected in the HEK293 cells transfected with empty vector (HEK293-CV cells). Expression of both OATP1B1 and OATP1B3 was enhanced by the treatment with 10mM sodium butyrate for 24 h; thus, we treated the cells with 10mM sodium butyrate before uptake study but not before accumulation study and other cytotoxicity study.
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Cytotoxicity of Microcystin-LR and Inhibition of its Cytotoxicity
To further evaluate the role of the transporters in microcystin-LR cytotoxicity, we examined the cytotoxic effect of microcystin-LR on HEK293-OATP1B1 and HEK293-OATP1B3 cells. HEK293-OATP1B1 (IC50 6.6nM) and HEK293-OATP1B3 cells (IC50 6.5nM) were equally very sensitive to microcystin-LR. In contrast, HEK293-CV cells were resistant to microcystin-LR up to a concentration of 200nM (data not shown). We further studied whether known substrates and inhibitors of OATP1B3 attenuate the microcystin-LR–induced cytotoxicity using HEK293-OATP1B3 cells. Table 1 summarized the effect of BSP, rifampicin, and CsA on the sensitivity of the cells to microcystin-LR. Rifampicin and CsA potently inhibited the cytotoxicity of microcystin-LR in HEK293-OATP1B3 cells in a dose-dependent manner. BSP, which is a relatively good substrate of OATP1B1 than that of OATP1B3, also weakly modulated the cytotoxicity. These agents used above did not affect the sensitivity of HEK293-CV cells to microcystin-LR (data not shown).
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Uptake of Microcystin-LR Mediated by OATP1B3 and Inhibition of its Uptake
We analyzed the uptake property of hepatotoxin microcystin-LR into the cells expressing the transporting carrier protein, OATP1B3. Uptake study was carried out under the serum-free conditions. We measured uptake of microcystin-LR in HEK293-OATP1B3 cells at various concentrations of microcystin-LR (1–10 µM) after 15 min incubation by ELISA assay (Fig. 2). Microcystin-LR transported into the HEK293-OATP1B3 cells was saturable, and the Km value was 1.2µM (Fig. 2A). We further examined the inhibitory effects of BSP, rifampicin, and CsA on the uptake of 1µM microcystin-LR. The selective uptake into the cells mediated by OATP1B3 was considerably inhibited by 25µM BSP and 10µM rifampicin. Two hundred nano molar of CsA effectively inhibited the uptake of microcystin-LR by approximately 23% (Fig. 2B).
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Accumulation of Microcystin-LR Mediated by OATP1B1 or OATP1B3
We analyzed the intracellular accumulation of hepatotoxin microcystin-LR after its selective uptake into the HEK293-OATP1B1 or HEK293-OATP1B3 cells to verify the toxic activity of the hepatotoxin microcystin-LR. Accumulation study was carried out under serum-containing conditions, and the accumulation was measured by ELISA assay (Fig. 3). Cells were incubated with several concentrations of microcystin-LR for 2 h at 37°C. The accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased according to the increased concentration of microcystin-LR in the medium. After incubation with 50nM microcystin-LR, 7.6 or 3.5 pg microcystin-LR/mg protein was accumulated in the HEK293-OATP1B1 or HEK293-OATP1B3 cells, respectively. In contrast, the accumulation of microcystin-LR in HEK293-CV cells was negligible (Fig. 3).
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Inhibition of PP2A Activity by Microcystin-LR
We measured the cellular serine/threonine PP activity in HEK293-OATP1B3 cells treated with microcystin-LR (Fig. 4), since the target of microcystin-LR is a mainly PP2A that is a member of the serine/threonine PP family. The total cellular serine/threonine PP activity was decreased by microcystin-LR in a dose-dependent manner. After incubation with 50 and 100nM of microcystin-LR for 6 h, the PP activity was decreased by approximately 20 and 30%, respectively. In contrast, the PP activity in the HEK293-CV cells was not affected by the microsystin-LR.
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Analysis of Microcystin-LR–Induced Apoptosis
To elucidate the mechanism of the cytotoxicity of microcystin-LR, we measured the proportion of apoptotic cells by FACS analysis. The early stage of apoptosis is characterized by plasma membrane alterations, which can be detected by Annexin V-fluorescein staining. We thus examined the apoptotic HEK293-OATP1B3 cells by staining with Annexin V-FITC and with PI. Flow cytometric analysis demonstrated that the proportion of apoptotic HEK293-OATP1B3 cells, which were positive for Annexin V but negative for PI after incubation with 10 and 50nM microcystin-LR for 24 h were 11.1 ± 1.5 % and 10.3 ± 4.6%, respectively, and approximately eightfold higher than that of control cells without microcystin-LR (Fig. 5A). The necrotic HEK293-OATP1B3 cell, which was positive for PI but negative for Annexin V, was not detected (data not shown).
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We also measured sub-G1 proportion of HEK293-OATP1B3 cells treated with 10 or 50nM microcystin-LR for 24 h. The sub-G1 proportion of HEK293-OATP1B3 cells treated with 50nM microcystin-LR was increased, and 10nM microcystin-LR was not sufficient to increase the sub-G1 proportion (Fig. 5B).
Activation of MAPKs by Microcystin-LR
We analyzed the microcystin-LR–induced activity of MAPKs such as ERK1/2, JNK, and p38, which are supposed to be the direct or indirect substrates of PP2A. ERK1/2, JNK, and p38 were slowly phosphorylated after incubation with 50nM microcystin-LR. JNK phosphorylation was considerably enhanced from 3.5 to 10 h after addition of 50nM microcystin-LR. A low level of ERK1/2 phosphorylation was detectable in untreated cells, and this was largely enhanced after 8 h incubation with microcystin-LR (Fig. 6). In contrast, the expression level of these MAPKs and -tubulin as an internal control was not varied by the microcystin-LR.
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Next, we analyzed the effect of the MAPK kinase inhibitor or MAPK inhibitors on cytotoxicity of microcystin-LR. Interestingly, we found that U0126, a specific inhibitor of MEK1/2, which is MAPK kinase upstream of ERK1/2, a JNK inhibitor SP600125, and a p38 inhibitor SB203580 dose dependently attenuated the cytotoxicity (Fig. 7).
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Effect of N-Acetyl-L-Cysteine on Cytotoxicity of the Microcystin-LR
We studied whether NAC, a ROS scavenger, attenuates cytotoxicity of microcystin-LR. HEK293-OATP1B3 cells were preincubated with various concentrations of microcystin-LR for 1 h, and then NAC was added in the medium. The cells were further incubated for another 3 days. The NAC specifically modulated cytotoxicity of low concentrations of microcystin-LR up to 20nM, and its DMFs were 0.7 and 0.4 at 1 and 2.5mM NAC, respectively. (Fig. 8)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The targets of microcystin-LR are serine/threonine PP, PP1 and PP2A. Microcystin-LR exclusively induces liver damage and/or promotes the hepatocarcinogenesis. In this study, we demonstrated that OATP1B1 and OATP1B3 mediate transport of intact microcystin-LR into the cells and induce their consequent cytotoxicity. Microcystin-LR was dose dependently accumulated in the HEK293-OATP1B1 and HEK293-OATP1B3 cells. In contrast, accumulation of microcystin-LR in HEK293-CV cells was not detected. These results indicated that the expression of OATP1B1 or OATP1B3 in HEK293 cells resulted in the intracellular accumulation of microcystin-LR.
Previous reports demonstrated that microcystin-LR was hardly cytotoxic to cultured cell lines (Botha et al., 2004; Chong, 2000; Lankoff et al., 2003, in press; Zhan et al., 2004; Zegura et al., in press). A few cell lines were responded to relatively high concentrations (micro molar orders) of microcystin-LR (Chong et al., 2000). In this study, we confirmed that low concentrations of microcystin-LR were not cytotoxic to HEK293, MDCKII, and HepG2 cell lines (data not shown). However, we found that transfection of OATP1B1 or OATP1B3 cDNA into HEK293 cells increased the sensitivity to microcystin-LR of HEK293 cells suggesting that both OATP1B1 and OATP1B3 can mediate transport of microcystin-LR into hepatocytes. This finding suggests that both OATP1B1 and OATP1B3 play a key role in the tissue-specific toxicity of microcystin-LR. Furthermore, we found an approximately two- to fourfold higher accumulation of microcystin-LR in HEK293-OATP1B1 cells than in HEK293-OATP1B3 cells. In spite of this difference, we found that microcystin-LR exerted a similar cytotoxicity in both cells. We suppose our finding suggests that a sufficient level of intracellular accumulation of microcystin-LR to induce cytotoxicity is low, and the level of microcystin-LR accumulated in HEK293-OATP1B3 cells was enough to induce cytotoxicity of microcystin-LR.
Substrates and inhibitors of OATP1B3 including rifampicin and CsA (Letschert et al., 2006; Vavricka et al., 2002) strongly attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR cytotoxicity. These findings confirm that the expression of OATP1B3 in the cells mediates the cytotoxicity of microcystin-LR. On the other hand, pigment BSP which is preferentially transported by OATP1B1 rather than OATP1B3 (Cui et al., 2001), slightly attenuated the cytotoxic effect of microcystin-LR on HEK293-OATP1B3 cells. This result suggests that BSP and microcystin-LR may be transported by OATP1B3 through different binding sites. In contrast to cytotoxicity study, these agents efficiently inhibited uptake of microcystin-LR into HEK293-OATP1B3 cells. Both rifampicin and BSP considerably inhibited uptake of microcystin-LR into HEK293-OATP1B3 cells under serum-free condition. Cui et al., (2000) reported that OATP1B3-mediated transport of BSP was inhibited by the serum. Our discrepant results between cytotoxicity study and uptake study may be caused by the different conditions in the presence or absence of serum. On the other hand, cytotoxicity of microcystin-LR was attenuated by CsA in HEK293-OATP1B3 cells. CsA is also a specific inhibitor of mitochondrial permeability transition in rat hepatocytes (Ding et al., 2002). Recently, Nakagawa et al. (2005) reported that mitochondrial permeability transition was not observed in CsA target CypD knock out mice. These reports suggest that CsA inhibited the microcystin-LR–induced apoptosis by suppressing OATP1B3 activity. Further study is needed to elucidate whether the inhibitory effect of CsA on mitochondrial permeability transition as well as transporting activity of OATP1B3 attenuated the cytotoxicity of microcystin-LR on HEK293-OATP1B3 cells.
Fischer et al. (2005) reported that OATP1B3 transports 3H-labeled dihydromicrocystin-LR with 9 ± 3µM Km value by using Xenopus oocyte transport system. However, we demonstrated the transport activity of intact microcystin-LR with 1.2µM of Km value for OATP1B3 by using HEK293-OATP1B3 cells, indicating transport activity from present study is higher than Fischer's result. We suppose this discrepancy may be due to the difference of the several experimental conditions; e.g., transport systems between Xenopus oocyte and mammalian cell line, substrates between dihydromicrocystin-LR and intact microcystin-LR, or assay systems between assay with radioactive substrate and ELISA.
We further demonstrated the molecular basis for the cytotoxicity of microcystin-LR after its selective uptake into the HEK293-OATP1B3 cells. We measured intracellular serine/threonine PP activity to confirm that microcystin-LR inhibits the PP1 and PP2A activity. Total intracellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR in a dose-dependent manner, and approximately 20 and 30% of the activity was decreased after 6 h incubation of the cells with 50 and 100nM of microcystin-LR, respectively, compared with control without microcystin-LR. In contrast, the influence of microcystin-LR to the HEK293-CV cells was negligible as expected.
We demonstrated that microcystin-LR induced apoptotic cell death but not necrosis. Although it is believed that caspases are common executers for apoptosis, activation of caspase-3 was negligible after exposure to 50nM microcystin-LR (data not shown). Our result is consistent with Ding's findings (2002) that activation of caspase-9 and -3 was not involved in apoptosis of rat hepatocytes induced by 1µM microcystin-LR.
Next, we analyzed phosphorylation of MAPKs such as ERK1/2, JNK, and p38 after incubation with microcystin-LR. ERK1/2, JNK, and p38 were considerably phosphorylated in a time-dependent manner in the presence of 50nM microsystin-LR. Phosphorylation of both ERK1/2 and p38 was considerably enhanced after 8 h incubation with 50nM microcystin-LR. JNK was highly phosphorylated after 3.5 h incubation under the same condition. Phosphorylation of these MAPKs was continuously increased up to at least 10 h incubation with 50nM of microcystin-LR. These results suggest that microcystin-LR disrupted the balance between protein phosphorylation and dephosphorylation. Zhu et al. (2005) reported that JNK and p38 but not ERK1/2 were phosphorylated in microcystin-LR–transformed the conditionally immortalized normal human colorectal crypt epithelial cell line NCC. We found that not only JNK and p38 but also ERK1/2 was substantially phosphorylated by microcystin-LR. This discrepancy may be due to the difference of experimental conditions, alternatively due to the difference of the cell types. In general, both JNK and p38 are activated by a variety of cellular stress including hypoxia, proinflammatory cytokines, UV, ROS, hyperosmolarity, and heat shock, and their activation is regulated by PP2A (Boudreau et al., 2007; Ikeda et al., 2006; Nebreda and Porras, 2000). On the other hand, ERK1/2 is activated in response to mitogen or growth factor stimulation (Fang and Richardson, 2005; Sasagawa et al., 2005). Interestingly, it has recently been reported that ERK1/2 activation is also associated with apoptosis signaling induced by cisplatin (Choi et al., 2004; Wang et al., 2000), betulinic acid (Rieber and Reber, 2006), deferoxamine (Lee et al., 2006), pifithrin- (Kaji et al., 2003), cadmium (Iryo et al., 2000), acrylamide (Okuno et al., 2005), and interferon-
(Horiuchi et al., 2006). These reports and our present results suggest that the activation of ERK1/2 might play an important role in the apoptosis induced by the microcystin-LR. The MAPK including ERK1/2, JNK, and p38 seemed to be closely related to apoptosis by microcystin-LR.
Plasma membrane–permeable PP1 and PP2A inhibitor, okadaic acid, also induces activation of ERK1/2 (Rossini et al., 1999), JNK (Yoon et al., 2006), and p38 (Boudreau et al., in press). Interestingly, ERK2 phosphorylation is dependent on cell type. No increase in ERK2 phosphorylation was observed in HeLa and The hepatoma cell line from Rattus novergicus cells exposed to okadaic acid (Rossini et al., 1999). Both microcystin-LR and okadaic acid are selective inhibitors of PP2A rather than PP1 (Chatfield and Eastman, 2004; Møller et al., 2004). PP2A exists as a stable core dimmer comprised of a catalytic subunit and one of at least 12 regulatory subunits. Depending on the combination of the catalytic subunit and regulatory subunit, the PP2A participate in a large variety of cellular activities via regulation of diverse signaling pathways (Boudreau et al., in press). Cellular balance between PP and kinase activity is a crucial regulatory mechanism for cellular activities, and PP2A plays an important role in maintaining the balance between cell survival and cell death (Boudreau et al., in press).
It is thought that cytotoxicity of relatively high concentration of microcystin-LR is caused by PP inhibition and generation of ROS. In this study, we analyzed the cytotoxic effect of relatively low concentration of microcytin-LR on HEK293-OATP1B3 cells. The cytotoxic effect of microcystin-LR at the range of 2–20nM was attenuated by ROS scavenger, NAC. The cytotoxic effect of microcystin-LR at 50nM or more, however, was not influenced by the NAC. These results suggest that ROS generated by microcystin-LR might have induced, at least in part, the cytotoxic effect of microcystin-LR in relatively low concentration ranges. At high concentrations of microcystin-LR, the cytotoxicity might be induced mainly through PP2A inhibition. Therefore, ROS might be associated with the cytotoxicity of microcystin-LR but probably to a lesser degree than PP2A inhibition and consequent MAPK activations. Further study is necessary to elucidate whether ROS generation by the relatively low dose of the microcystin-LR is involved in the apoptosis.
In conclusion, the present study demonstrated that OATP1B3-mediated uptake of microcystin-LR induced apoptosis, indicating OATP1B3 as well as OATP1B1 is indispensable for the hepatocytotoxicity of microcystin-LR. Microcystin-LR–induced apoptosis was mainly mediated by the inhibition of the PP2A and consequent activation of MAPKs. Therefore, both HEK293-OATP1B1 and HEK293-OATP1B3 cells are quite useful tools for the study on the cytotoxicity of microcystin-LR.
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ACKNOWLEDGMENTS |
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We express great appreciation to Dr Wolfgang Hagmann for encouragements. We also thank Chiko Yumiba for excellent secretarial assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Wakate (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (# 17790369). Conflicts of interest: None declared.
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