Cadmium induces autophagy via IRE1 signaling pathway activated by Ca2 + in GC-2spd cells

Abstract

Cadmium (Cd), an environmental toxicant, accumulates in the human body and damages the male reproductive system. To investigate the molecular mechanisms underlying Cd-induced reproductive toxicity, we used GC-2spd cells and treated them with CdCl₂. Additionally, we added 2-APB (an inhibitor of the IP3R) and STF-083010 (an inhibitor of IRE1) to investigate whether they could ameliorate Cd-induced reproductive toxicity. Confocal microscopy and flow cytometry confirmed that CdCl₂-treated GC-2spd cells displayed imbalance of calcium homeostasis, with upregulation of the expression of the IP3R, a key pathway for endoplasmic reticulum (ER) Ca²⁺ release. Furthermore, the ER stress (ERS) effector protein IRE1 expression was also increased, suggesting that Cd activated ERS and the IRE1 pathway by disrupting calcium homeostasis. Previous studies have shown that ERS induces autophagy. We performed the MDC assay to detect autophagosome formation, revealing increased expression of autophagy-related proteins LC3-II/LC3-I and Beclin-1 in response to Cd treatment. In contrast, treatment with 2-APB and STF-083010 inhibited autophagy and mitigated cell death. This inhibitory effect may be due to 2-APB blocking IP3R-mediated Ca²⁺ release, alleviating imbalance of calcium homeostasis, while STF-083010 inhibits IRE1, restoring ER homeostasis and reducing autophagy. These findings suggest that imbalance of calcium homeostasis activates the IRE1 pathway-mediated ERS, leading to excessive autophagy and male reproductive toxicity. Conversely, the addition of 2-APB and STF-083010 reversed these effects, synergistically restoring intracellular Ca²⁺ homeostasis and inhibiting ERS to promote cell health. This study provides a new therapeutic strategy for Cd-induced male reproductive disorders.