Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3.
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 (IC(50) 6.6nM) and HEK293-OATP1B3 cells (IC(50) 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 1.2 microM 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.
Komatsu
Masaharu
M
Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. haruto@m3.kufm.kagoshima-u.ac.jp
Furukawa
Tatsuhiko
T
Ikeda
Ryuji
R
Takumi
Shota
S
Nong
Qingqing
Q
Aoyama
Kohji
K
Akiyama
Shin-Ichi
S
Keppler
Dietrich
D
Takeuchi
Toru
T
D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000633:toxicity; D005434:Flow Cytometry; D006801:Humans; D007202:Indicators and Reagents; D027381:Liver-Specific Organic Anion Transporter 1; D008387:Marine Toxins; D052998:Microcystins / Q000378:metabolism* / Q000633:toxicity*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D027361:Organic Anion Transporters / Q000502:physiology*; D029362:Organic Anion Transporters, Sodium-Independent / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D027381:Liver-Specific Organic Anion Transporter 1; D008387:Marine Toxins; D052998:Microcystins; D027361:Organic Anion Transporters; D029362:Organic Anion Transporters, Sodium-Independent; C503999:SLCO1B1 protein, human; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D020928:Mitogen-Activated Protein Kinases; C057862:cyanoginosin LR; D000111:Acetylcysteine