Alterations of tau and VASP during microcystin-LR-induced cytoskeletal reorganization in a human liver cell line.

Previously, we have reported alterations to HSP27 during Microcystin-LR (MC-LR)-induced cytoskeletal reorganization in the human liver cell line HL7702. To further elucidate the detailed mechanism of MC-LR-induced cytoskeletal assembly, we focused on two cytoskeletal-related proteins, Tau and VASP. These two proteins phosphorylated status influences their ability to bind and stabilize cytoskeleton. We found that MC-LR markedly increased the level of Tau phosphorylation with the dissociation of phosphorylated Tau from the cytoskeleton. Furthermore, the phosphorylation of Tau induced by MC-LR was suppressed by an activator of PP2A and by an inhibitor of p38 MAPK. VASP was also hyperphosphorylated upon MC-LR exposure; however, its phosphorylation appeared to regulate its cellular localization rather than cytoskeletal dynamics, and its phosphorylation was unaffected by the PP2A activator. These data suggest that phosphorylated Tau is regulated by p38 MAPK, possibly as a consequence of PP2A inhibition. Tau hyperphosphorylation is likely an important factor leading to the cytoskeletal destabilization triggered by MC-LR and the role of VASP alteration upon MC-LR exposure needs to be studied further. To our knowledge, the finding that Tau is implicated in cytoskeletal destabilization in MC-LR-treated hepatocytes and MC-LR-induced VASP's alteration has not been reported previously.

Microcystin-LR induces protein phosphatase 2A alteration in a human liver cell line.

Microcystin-LR (MC-LR) is a potent inhibitor of protein phosphatases 1 and 2A, and has potent hepatotoxicity and tumor promotion activity. Numerous studies on MC-LR toxicity have been conducted in rat hepatocytes, but few studies of the effects of microcystins on human hepatocytes have been done. In this study, HL7702 cells (a human normal liver cell line) were incubated in MC-LR for 24 h. The existence of MC-LR in HL7702 cells was confirmed. Furthermore, PP2A activity and the alteration of PP2A subunits were assessed. The results show that PP2A activity decreased from the concentration of 1 µM MC-LR, showing a concentration-dependent decline, to about 34% at 10 µM MC-LR. This activity undergone opposite change with alternations of phosphorylated Y307-PP2A/C and PP2A/C subunit but showed same change with the alteration of the ratio of methylated L309-PP2A/C to PP2A/C. B55α, a regulatory subunit of PP2A, was slightly increases in cells treated with the highest concentration of MC-LR (10 µM), and colocalized increasedly with rearranged-microtubules after 1 µM MC-LR exposure. However, the proportion of early apoptotic cells did not show any change at various concentration of MC-LR for 24 h. To our knowledge, this is the first report showing MC-LR-induced alteration of PP2A phosphatase in human cultured hepatocytes, and the mechanism of action seems to be similar as described before in vitro. The alteration of PP2A and microtubule seems to be the early event induced by MC-LR exposure.

Effect of microcystin-LR on protein phosphatase 2A and its function in human amniotic epithelial cells.

Due to their toxicity, the increased distribution of microcystins (MCs) has become an important worldwide problem. MCs have been recognized as inhibitors of protein phosphatase 2A (PP2A) through their binding to the PP2A catalytic subunit. However, the exact mechanism of MC toxicity has not been elucidated, especially concerning the cellular response and its autoregulation. To further dissect the role of PP2A in MC-induced toxicity, the present study was undertaken to determine the response of PP2A in human amniotic epithelial (FL) cells treated with microcystin-LR (MCLR), one of the MC congeners. The results show that a low-dose treatment of MCLR in FL cells for 6 h induced an increase in PP2A activity, and a high-dose treatment of MCLR for 24 h decreased the activity of PP2A, as expected. The increased mRNA and protein levels of the PP2A C subunit may explain the increased activity of PP2A. Furthermore, MCLR altered microtubule post-translational modifications through PP2A. These results further clarify the underlying mechanism how MCLR affects PP2A and may be helpful for elucidating the complex toxicity of MCLR.

Regulation of heat shock protein 27 phosphorylation during microcystin-LR-induced cytoskeletal reorganization in a human liver cell line.

Acute exposure to microcystin-LR (MC-LR) can induce the reorganization or disruption of the cytoskeleton, but proteins or enzymes correlated with this stress response have not been fully identified. Here, we report alterations to HSP27 during MC-LR-induced cytoskeletal reorganization in the human liver cell line HL7702. The cells incubated with MC-LR exhibited the rearrangement of filamentous actins and microtubules. The activity of protein phosphatase 2A was greatly decreased by MC-LR exposure. Furthermore, MC-LR markedly increased the level of HSP27 phosphorylation with the enhanced distribution of phosphorylated HSP27 to the cytoskeleton. To further determine the regulation of MC-LR-induced HSP27 phosphorylation, the activation of the MAPK superfamily was assessed. The result showed phospho-activation of p38 MAPK, JNK and ERK1/2 by MC-LR. Increases in HSP27 phosphorylation were suppressed by pretreating cells with SB203580 or SP600125, which are inhibitors of p38 MAPK or JNK, respectively. These data suggest that phosphorylated HSP27 is involved in cytoskeletal reorganization and is regulated by MAPKs, possibly as a consequence of PP2A inhibition. Moreover, the regulation of HSP27 phosphorylation may be important in MC-LR-induced cytoskeleton reassembly, which may provide helpful insights into the mechanism of MC-LR toxicity.