Microcystin-LR Promotes Liver Cells Proliferation By Activating Akt/mTORC1/S6K1 Pathway

BackgroundMicrocystins (MCs) are a group of cyclic heptapeptides, produced by blue-green algae, which are secreted into water during cyanobacterial blooms and have been demonstrated to pose a health threat to humans through the food chain. Therefore, the research about MCs has been a hotspot of environmental science and preventive medicine in the last three decades. More than 90 different structural analogues of microcystins have been reported. Among them, microcystin-LR (MC-LR) is the most common and toxic variant, and the liver is its main target organ. It has been demonstrated that MC-LR can induce serious hepatotoxicity, including cell apoptosis, necrosis, hepatomegaly, pooling of blood, and liver damage. Additionally, the epidemiological studies indicate that MC-LR is a tumor promoter and closely related to a high incidence of primary liver cancer (PLC). In general, the main toxic mechanism of MC-LR is its potent inhibition on protein phosphatase 2A (PP2A), then leading to various cytological effects and pathological changes, which is also the key to uncover MC-LR-induced toxicity. Our previous work have demonstrated that MC-LR can accumulate in various cell lines and inhibit the total PP2A activity at 24 h exposure, which increase the phosphorylation of various PP2A substrates and induce the cytoskeleton rearrangement. Nevertheless, no toxic significant cellular effects were observed.ObjectivePP2A has been demonstrated to be involved in multiple cellular signaling and diverse physiological processes. Sustained dysfunction of such an important enzyme by MC-LR certainly has some kind of effects on cellular processes besides the molecular changes. Therefore, prolonged MC-LR exposure was designed to further study the effects of MC-LR to PP2A substrates and the related signaling pathways as well as the physiological processes. And, the experiments in vivo were also performed to confirm the findings in vitro.MethodsHL7702 cells were exposed to 1-10 μM MC-LR for 1-72 h to explore the effects of MC-LR to PP2A activity, cell proliferation, cell apoptosis and cell cycle as well as the related mechanisms. Meanwhile, ICR mice injected intraperitoneally with 20-80 μg/kg MC-LR for 2 h-4 d were used to confirm the results in vitro.ResultsExperiments in vitro:1. MC-LR accumulated in HL7702 cells and bound to PP2A/C within 1 h exposure, which did not increase with the exposure time.2. MC-LR caused concentration-dependent inhibition on the total PP2A activity in HL7702 cells, which maintained at a lower level during 24-72 h exposure.3. MC-LR induced PP2A regulator a4 to release its reserved PP2A/C.4. MC-LR promoted HL7702 cells proliferation significantly since 36 h exposure.5. MC-LR did not affect HL7702 cell apoptosis and cell cycle within 48 h exposure.6. MC-LR activated Akt/mTORC1/S6K1 pathway since 1 h exposure and both the activated-Akt and the inhibited-PP2A are responsible for the S6K1 activation.7. Akt inhibitor blocked MC-LR-promoted cell proliferation.8. MC-LR increased the phosphorylation levels of Bcl-2 and Bad since 1 h exposure.9. MC-LR activated Cdkl (cyclin-dependent protein kinase 1) since 1 h exposure. Experiments in vivo:1. MC-LR accumulated in mice liver cells and bound to PP2A/C within 2 h exposure, which did not increase with the exposure time.2. The inhibition of MC-LR to PP2A activity in mice liver was related to the exposure concentration and time.3. MC-LR induced PP2A regulator a4 to release its reserved PP2A/C in mice liver.4. MC-LR induced liver cells proliferation.5. MC-LR activated Akt/mTORCl/S6K1 and Akt/β-catenin pathways in mice liver.6. It was found that MC-LR activated ERK/JNK/p38 in HL7702 cells in other study of our lab. In this research, we demonstrated MC-LR could also activate ERK/JNK/p38 as well as their downstream transcriptional factors in mice liver.ConclusionThe conclusions based on the results in vitro and in vivo are shown as below:1. MC-LR can accumulate in liver cells and bind to PP2A/C within a very short time, which subsequently inhibits PP2A activity dependent on exposure concentration and time.2. PP2A regulator α4 slows the decline of PP2A activity caused by MC-LR through releasing its reserved PP2A/C.3. MC-LR exposure promotes liver cell proliferation and the activation of Akt/mTORC1/S6K1 due to the inhibition of PP2A is the main mechanism. And, the activated Akt/β-catenin and ERK/JNK/p38 MAPK may be also involved in that process.4. Although MC-LR disorders the levels of apoptosis-and cell-cycle-related proteins, which are not sufficient to induce the changes of the related cellular processes.