Cellular Regulation Of PP2A And Its Effect On Apoptotic Fate Of HEK293Cells Under Microcystin-LR Exposure

Eutrophication and blue alga bloom in waters are causing serious environmental disasters worldwide. One group of blue alga, microcystis, produces microcystins (MCs) in water bloom that pose severe hazard to human health due to their strong toxicity. Among them, microcystin-LR (MCLR) has hepatoxicity, nephrotoxicity and neurotoxicity, and is one of the most toxic member of MCs. Studies have revealed that MCLR can cause cell death, while can also promote carcinogenesis. However, under exposure of MCLR, the determinant factors of cell fate are not clear.Protein phosphatase2A (PP2A) is a main target of MCLR. PP2A has essentially important functions in cells and is involved in almost all the cellular activities, such as cell proliferation, metabolism, differentiation, transformation, DNA repair and apoptosis. PP2A holoenzyme is comprised of a scaffold subunit (PP2A/A), a catalytic subunit (PP2A/C), and one of many regulatory subunits (PP2A/B). The regulatory subunit determines the substrates, sub-cellular localization and the function of PP2A holoenzyme. At least75kinds of PP2A holoenzymes have been found and each has a variety of substrates. Other than the classical combination, a small part of PP2A/C binds to a4protein and has lower activity than the other forms. However, this form of combination plays an important role in cellular regulation of PP2A activity when under stress. Previous studies have found that the direct binding of MCLR to PP2A/C inhibits its phosphatase activity. This feature of MCLR has long been considered the major mechanism by which MCLR poses cytotoxicity. However, apart from the direct binding and thus inhibition of PP2A activity, other effects of MCLR on cellular PP2A holoenzymes are largely unknown. Moreover, because of the essential importance of PP2A, its activity is under tight regulation by the cells. So far, it has been found that the cells regulate PP2A activity by phosphorylation and methylation of PP2A/C, by generating ceramide which upregulates certain forms of PP2A holoenzyme (CAPP, ceramide activated protein phosphatase), as well as by dissociation of PP2A/C and α4protein. Therefore we ask, when under MCLR insult, by which mechanisms cells may regulate PP2A activity; is the regulatory effect enough to compensate the activity loss caused by MCLR; and what are the consequential cellular effects?Previous proteomic studies have shown that under the exposure of MC, a large spectrum of signal proteins are altered. Importantly, most of those proteins are related to PP2A. Thus, we hypothesize that under the exposure of MCLR, the activity of PP2A, especially the activity of its holoenzyme, as well as the regulation of PP2A from the cells, play an important role in the fate determination of the cells. The present study utilizes Human Embryonic Kidney293(HEK293) cell line, to study the PP2A subunit and activity change, the phosphorylation of its substrates, the regulation of PP2A activity, the morphological change of cytoskeleton and focal adhesion, and cell fate, at the time when cells are treated with MCLR at concentrations that do not cause massive cell death. This study also uses mouse kidney as in vivo subject to reinforce some in vitro result.Major findings1. MCLR can directly bind to PP2A/C in HEK293cells. The concentrations of MCLR used in this study do not cause massive cell death, though tend to inhibit their viability.2. MCLR does not affect the protein levels of PP2A/A, PP2A/C and PP2A/B56δ in HEK293cells, but upregulates the protein levels of PP2A/B55a and PP2A/B56a; MCLR downregulates the methylation and ubiquitination of PP2A/C, but does not affect its phosphorylation; MCLR causes PP2A/C dissociate from its ubiquitinligase Mid1; relative higher concentration of MCLR causes dissociation of PP2A/A and PP2A/C; MCLR causes PP2A/B55a locate to Golgi apparatus but not PP2A/B56a; MCLR causes the dissociation of PP2A/C and a4protein, and a4protein locate to nucleus, but not PP2A/C.3. Relatively low concentrations of MCLR upregulate, while relatively high concentrations of MCLR inhibit cellular PP2A activity.4. MCLR induces the generation of ceramide. Using desipramine (DESI) to inhibit the generation of ceramide can alleviate the upregulation of PP2A/B55a and PP2A/B56α on protein level, and the upregualtion of PP2A activity at relatively low MCLR concentration, while aggravate the inhibition of PP2A by relatively high concentration of MCLR treatment, till almost complete inhibition.5. MCLR decreases of phosphorylation of c-Myc, a substrate of PP2A holoenzyme containing PP2A/B56a, while does not affect c-Myc protein level. MCLR upregulates the protein level, and decreases of phosphorylation ratio of Bad, another substrate of PP2A holoenzyme containing PP2A/B56a. MCLR also downregulates Bcl-2on protein level, but does not affect Bax. Under DESI co-treatment, the effects of MCLR on Bad and Bcl-2are abated. MCLR also upregulates the phosphorylation of p38MAPK and JNK.6. MCLR changes the morphology of HEK293cells. MCLR causes the depolymerization of cell filamentsactin, and the contraction of vimentin and microtubulin. The phenotype is similar to cells treated only with C6-cereamide. The disruption of cytoskeleton is alleviated when co-treated with MCLR and DESI. MCLR also causes cytoskeleton-related proteins Racl and Midi locate to nucleus.7. MCLR changes the morphology of viculin and disrupts the cell attachment to culture matrix. MCLR causes condensation and fragmentation of nuclei, indicating apoptosis. These effects are aborted when co-treated with DESI.8. Ceramide generation and increased apoptosis can be found in mouse kidney after MCLR injection. Major conclusionBesides that MCLR can directly bind to PP2A in HEK293cells, MCLR can also stimulate regulatory of PP2A in the cells, including the generation of ceramide, as well as dissociation of PP2A/C and a4. Furthermore, MCLR affects the cytoskeleton stability, cell attachment and induces apoptosis of HEK293cells. These cellular effects are related with ceramide, and also consistent with the loss of a4function due to dissociation with PP2A/C. Moreover, MCLR can induce ceramide generation and apoptosis in mouse kidney. Therefore, the results suggest that the holoenzyme activity of PP2A, and cellularregulation of PP2A, may be essential for the apoptotic fate of HEK293cells under MCLR exposure.