IRE1 and CaMKKβ pathways to reveal the mechanism involved in microcystin-LR-induced autophagy in mouse ovarian cells

Microcystin-LR (MC-LR) is an emerging water pollutant produced by blooming cyanobacteria. It could be absorbed into human body via contaminated food and drinking water causing severe reproductive toxicity. Previous studies showed that MC-LR could regulate autophagy by inducing endoplasmic reticulum (ER) stress thereby causing female reproductive toxicity. However, the molecular mechanisms of MC-LR-induced autophagy remain to be elucidated. It is known that IRE1 and CaMKK beta pathways are two important pathways involved in autophagy induced by ER stress. Hence, this study investigated the roles of both pathways in MC-LR-induced autophagy in mouse ovarian cells. The results showed that MC-LR significantly up-regulated the expression of autophagy marker proteins LC3II and BECLIN1 and down-regulated the expression of P62 in vivo and in vitro. MC-LR-caused increase of autophagosomes could be observed in KK-1 cells by MDC staining. MC-LR induced the formation of autolysosomes as indicated by the overlap of LAMP1 and LC3. Meanwhile, MC-LR significantly activated the proteins in IRE1 pathway (IRE1, XBP1 and JNK) and in CaMKKp pathway (CaMKK beta, AMPK, mTOR). Furthermore, MC-LR caused weight loss and ovarian histopathological damage in mice. In contrast, after the expression and function of IRE1 and CaMKKp were inhibited with siRNA in vitro and by inhibitors (4p8C and STO-609, respectively) in vivo, the up-regulation of LC3II and BECLIN1 and the degradation of P62 induced by MC-LR were significantly suppressed. MC-LR-induced autophagosomes in KK-1 cells and autolysosomes in mouse ovarian cells were also decreased. Moreover, the knockdown of IRE1 and CaMKK beta relieved MC-LR-induced histopathological injury to mouse ovaries. These results indicated that MC-LR induced ovarian cell autophagy and ovarian injury via IRE1 and CaMKK beta pathways. This study is the first study revealing the molecular mechanisms of MC-LR-induced autophagy of ovarian cells and providing new insights into the female reproductive toxicity of MC-LR.

Automated summary

MC-LR induces autophagy in ovarian cells and ovarian injury via the IRE1 and CaMKK beta pathways. Inhibition of these pathways can significantly alleviate autophagy and damage induced by MC-LR.