p53 Plays an important role in cell fate determination after exposure to microcystin-LR.
BACKGROUND
Microcystin-LR, a cyclic heptapeptide, possesses the ability to inhibit the serine/threonine protein phosphatases PP1 and PP2A and, consequently, exhibits acute hepatocytotoxicity. Moreover, microcystin-LR induces cellular proliferation, resulting in tumor-promoting activity in hepatocytes. However, mechanisms that regulate the balance between cell death and proliferation after microcystin-LR treatment remain unclear.


OBJECTIVE
We examined the contribution of the transcription factor p53, as well as that of the hepatic uptake transporter for microcystin-LR, organic anion transporting polypeptide 1B3 (OATP1B3), to the cellular response to microcystin-LR exposure.


METHODS
We analyzed intracellular signaling responses to microcystin-LR by immunoblotting and real-time reverse-transcriptase polymerase chain reaction techniques using HEK293 human embryonic kidney cells stably transfected with SLCO1B3 (HEK293-OATP1B3). In addition, we analyzed the effect of attenuation of p53 function, via the p53 inhibitor pifithrin-alpha, and knockdown of p53 mRNA on the cytotoxicity of microcystin-LR using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.


RESULTS
Microcystin-LR induced the phosphorylation and accumulation of p53 in HEK293-OATP1B3 cells, which resulted in up-regulation of the expression of p53 transcript targets, including p21 and seven in absentia homolog 1 (siah-1). In addition, microcystin-LR activated Akt signaling through the phosphorylation of Akt and glycogen synthase kinase 3beta. Although Akt signaling was activated, the accumulation of p53 led cells to apoptosis after treatment with 50 nM microcystin-LR for 24 hr. Both pharmacological inhibition of transcription factor activity of p53 by pifithrin-alpha and knockdown of p53 with small hairpin RNA attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR.


CONCLUSIONS
This study demonstrates the importance of p53 in the regulation of cell fate after exposure to microcystin-LR. Our results suggest that, under conditions of p53 inactivation (including p53 mutation), chronic exposure to low doses of microcystin-LR may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.
Takumi
Shota
S
Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Komatsu
Masaharu
M
Furukawa
Tatsuhiko
T
Ikeda
Ryuji
R
Sumizawa
Tomoyuki
T
Akenaga
Hitomi
H
Maeda
Yuta
Y
Aoyama
Kohji
K
Arizono
Koji
K
Ando
Seiichi
S
Takeuchi
Toru
T
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D052160:Benzothiazoles / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D008387:Marine Toxins; D052998:Microcystins / Q000633:toxicity*; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D052160:Benzothiazoles; D008387:Marine Toxins; D052998:Microcystins; D029362:Organic Anion Transporters, Sodium-Independent; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D016159:Tumor Suppressor Protein p53; D014050:Toluene; C121565:pifithrin; D051057:Proto-Oncogene Proteins c-akt; C057862:cyanoginosin LR