Microcystin-LR activates the ERK1/2 kinases and stimulates the proliferation of the monkey kidney-derived cell line Vero-E6

Abstract

a b s t r a c tMicrocystin-LR (MCLR) is a peptide produced by freshwater cyanobacteria that induces severe hepatotoxicity in humans and animals. MCLR is also a potent tumour promoter and it has been proposed that this activity is mediated by the inhibition of protein phosphatases PP1/PP2A, possibly through the activation of proto-oncogenes c-jun, c-fos and c-myc. However, the mechanisms underlying MCLR-induced tumour promotion are still largely unknown, particularly in non-liver cells. In previous studies we have demonstrated that micromolar concentrations of MCLR induce cytotoxic effects in the kidney Vero-E6 cell line. The purpose of the present work was to evaluate whether the exposure to subcytotoxic concentrations of MCLR was sufficient to induce the proliferation of Vero-E6 cells. Through BrdU incorporation assay we show that at nanomolar concentrations MCLR stimulates cell cycle progression in Vero-E6 kidney cell line. Moreover, the analysis of mitogen-activated protein kinases p38, JNK and ERK1/2 activity revealed that the proliferative effect of MCLR is associated with the activation of the pro-proliferative ERK1/2 pathway. These results emphasise the importance to confirm in vivo the impact of MCLR on tumour promotion at kidney level.

A review of reproductive toxicity of microcystins

“…Recent reports suggest that MCs modulate PPs activity not only by the direct inhibition of enzyme activity, but also through the regulation of protein expression [16][17][18][19][94][95][96][97]. PP1 and PP2A inhibition induces the disruption of the dynamic equilibrium of protein phosphorylation/dephosphorylation, leading to the damage of numerous cellular processes such as cytoskeleton organization, Wnt, Akt, p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) of mitogen-activated protein kinase (MAPK) signaling pathways, metabolism and cell cycle [20,[98][99][100][101] (Fig. 4).…”

The Kidney Vero-E6 Cell Line: A Suitable Model to Study the Toxicity of Microcystins

“…Few studies support this hypothesis: (1) Li et al [68] reported the activation of proto-oncogenes c-jun, c-fos and c-myc by a cyanobacterial extract containing microcystins in rat liver, kidney and testis; (2) Zhu et al [69] demonstrated that MCLR induces the transformation of immortalized colorectal crypt cells through the constitutive activation of AKT and MAPK (p38 and JNK) cascades. Our team has evaluated the effect of MCLR in Vero-E6 cell line proliferation through the BrdU incorporation assay that evaluates the G1/S transition in cell cycle [28]. We showed that MCLR (1 to 10 nM) induces a significant increase in Vero cells proliferation with a maximum of 2.2 fold increase at 5 nM [28].…”

“…Our team has evaluated the effect of MCLR in Vero-E6 cell line proliferation through the BrdU incorporation assay that evaluates the G1/S transition in cell cycle [28]. We showed that MCLR (1 to 10 nM) induces a significant increase in Vero cells proliferation with a maximum of 2.2 fold increase at 5 nM [28]. We further analyzed the expression of MAPK (ERK1/2, JNK and p38) by Western-blot and concluded that MCLR stimulates Vero cells proliferation by the activation of the ERK1/2 signaling pathway [28].…”

“…Surprisingly, we observed that the dose-response curve of cell viability decrease induced by MCLR was quite similar in Vero-E6 and in liver-derived cell lines [26]. Since then, we have been demonstrated that MCLR induces a multiplicity of effects on Vero cells within a wide range of concentrations [26][27][28][29][30], according to a dose-effect relationship ( Figure 3). …”

Microcystin-LR stabilizes c-myc protein by inhibiting protein phosphatase 2A in HEK293 cells

“…Unlike c-myc, PP2A is generally regarded as a tumor suppressor (Janssens et al, 2005). Several studies have shown that PP2A might be able to affect the phosphorylation status of threonine 58 and serine 62 of c-myc in the direct or indirect pathways (Arnold and Sears, 2008;Dias et al, 2010;Henriksson et al, 1993;Lutterbach and Hann, 1994;Pulverer et al, 1994;Takumi et al, 2010). So, theoretically, given its potent effect on PP2A activity, MC-LR might be able to regulate c-myc protein stability.…”