Effect Of Microcystin-LR On Protein Phosphatase 2A In Human Amnion Epithelia Cell Line And Research On Endo-Porter As The Transporter Of Microcystin-LR

Microcystins (MCs) are toxins produced by freshwater cyanobacteria that belong to the genera Micocystis, Anabaena, Nodularia, Oscillatoria, and Nostoc. MCs are too stable to degrade naturally and it is difficult to remove them from water. MCs are of increasing importance due to their toxicity and potent tumor promoting activity, so the exact mechanisms of MCs-induced toxicity need further investigation. MCs have been recognized as a family of monocyclic heptapeptide hepatotoxins. Two of the amino acids are variable. Till now, over 80 structural variants have been recognized. Microcystin-LR (MCLR) is the most frequently studied as well as the most toxic in the group of MCs, L and R meaning leucine (L) and arginine(R) respectively. PP2A is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function, such as metabolism, proliferation, differentiation and apoptosis. PP2A holoenzymes are heterotrimers consisting of a core dimmer, AC, and a regulatory B-type subunit. MCLR has been recognized as the strong inhibitor of PP2A, which may be one reason of the implicated intoxication mechanism of MCLR. Studies in our laboratory have revealed that exposure to MCLR, MCRR and bloom extract could increase the protein levels of the A subunit of PP2A. Therefore, the present study was designed to illustrate the precise events happening in MCLR-induced toxicity in particular concerning with PP2A. Furthermore, the liver is the prime target organ affected by MCLR. A group of hepatocyte uptake transporters termed organic anion transporting polypeptides (OATPs) present mainly in liver are crucial for MCLR transferring into cells. In addition, it should be of great concern that accumulation of MCs in other organs without expression of OATPs has also been reported in recent studies. Therefore, to investigate the mechanism of MCLR toxicity in other cells without/low OATPs expression, we testified an alternative approach to study the toxicity of MCLR using the facilitated transporter Endo-Porter (EP)-a peptide reagent binding to cell membrane.The present study was focused on the binding of MCLR to PP2Ac, the effects of MCLR on PP2A activity and subunits, the regulation of PP2A on the post-translational modification of microtubule after MCLR treatment and the cytotoxicity of MCLR in the presence of Endo-porter in FL cells. MTT was used to evaluate cell viability; real-time PCR and western blot were used to detect the expression level of mRNA and protein respectively; cell cycle and apoptosis were measured by PI staining and Annexin V-PI staining; immunofluorescence was used to detect the co-locolization of aimed proteins and morphology of microtubule.The results were shown as below:1. MCLR treatment increased the activity of PP2A as well as the expression levels of mRNA and protein of PP2Ac, decreased the phosphorylation and methylation of PP2Ac.2. Exposure to MCLR had no effect on FL cells morphology and protein level of a tubulin. However, the binding of B55a and tryosinated tubulin was significantly reduced after MCLR treatment.3. MCLR exposure changed the levels of mRNA of PP2A regulatory subunits, however, exposure to MCLR had no significant effects on the protein levels.4. The accumulation of MCLR in FL cells in the presence of EP induced toxic effects including decrease of PP2A activity, inhibition of cell viability, increase of apoptotic rate, and increased generation of intracellular ROS and the elevation of Bax/Bcl-2 ratio.5. The present study revealed that MCLR could activate ERK1/2, JNK and p38. Furthermore, MCLR activated MEK1/2-ERK1/2-Myc pathway in a time-dependent manner. 6. MCLR exposure in the presence of EP induced cell cycle arrest and up-regulation of phosphrylated cdc25C at Ser216.The conclusions were shown as below:1. It is supposed that the hormesis response induced by MCLR increased the expression levels of mRNA and protein of PP2Ac, resulting in the increased activity of PP2A.2. The changed levels of mRNA of PP2A regulatory subunits by MCLR treatment suggest that the expression level of mRNA is more sensitive to MCLR treatment than that of protein.3. Exposure to MCLR influences the regulatory role of PP2A-B55αon tryosinated tubulin.4. The toxic effects of MCLR in FL cells in the presence of EP suggest that the utility of EP might be of great help for elucidating the complicated toxicity of MCLR in vitro.5. The ERK1/2 pathway might play an important role in the apoptosis induced by MCLR in the presence of EP.6. The up-regulation of phosphrylated cdc25C at Ser216 could contribute to the cell cycle arrest induced by MCLR in the presence of EP.