ZEA-induced autophagy in TM4 cells was mediated by the release of Ca2+ activates CaMKKβ-AMPK signaling pathway in the endoplasmic reticulum

Abstract

Zearalenone (ZEA), a prevalent non-steroidal estrogenic mycotoxin, is primarily produced by Fusarium contamination. Previous research indicated that ZEA induces autophagy in Sertoli cells (SCs), but the precise mechanisms underlying this process remain unclear. Several studies have suggested that elevated levels of cytoplasmic calcium ions (Ca2+) can trigger autophagy through the CaMKKβ and AMPK pathways. Therefore, this study investigated the potential mechanism by examining the activity of the calmodulin-dependent kinase kinase β (CaMKKβ) and AMP-activated protein kinase (AMPK) signaling pathway in TM4 cells exposed to ZEA. The findings of this study demonstrate that ZEA activates both the CaMKKβ and AMPK signaling pathways. Notably, the effects of ZEA on AMPK, the conversion of LC3I to LC3II, and the distribution of LC3 puncta were significantly inhibited by an AMPK inhibitor and stimulated by an AMPK activator. Furthermore, the concentration of cytosolic calcium (Ca2+) gradually increased with increasing concentrations of ZEA. Treatment of ZEA-exposed cells with the calcium chelator BAPTA-AM and the IP3 receptor antagonist 2-APB resulted in a significant reduction in intracellular Ca2+ concentration. This reduction was accompanied by decreased activities of CaMKKβ and AMPK, as well as a subsequent decrease in autophagy. Moreover, the antioxidant NAC significantly reduced the activities of AMPK and autophagy-related proteins. Based on these observations, it can be proposed that ZEA-induced ROS-mediated ER stress activates AMPK via Ca2+-CaMKKβ, ultimately leading to autophagy in TM4 cells.

1. Introduction

Zearalenone (ZEA) is a non-steroidal mycotoxin known for its estrogenic effects. It is produced by various Fusarium species through the polyketide pathway and is also referred to as the F-2 toxin. This mycotoxin is commonly found in contaminated grains such as corn, wheat, sorghum, and rice. Due to its acute and chronic toxicity to both humans and animals, ZEA poses a significant global health concern. Research has shown that ZEA can accumulate within the body, leading to toxic effects on the reproductive and immune systems. Notably, the reproductive system is particularly vulnerable to ZEA toxicity. Numerous animal studies have demonstrated that ZEA binds to estrogen receptors and alters the reproductive tract, consequently modifying reproductive behaviors in both animals and humans. Furthermore, ZEA has been linked to decreased fertility, reduced litter size, and lower sperm counts. Some studies indicate that ZEA can reduce the number of germ cells in male animals and impair sperm motility. Even low doses of ZEA can affect sperm production and semen quality. For instance, male CD-1 mice treated with 20 and 40 μg/kg of ZEA exhibited a significant decrease in the number of spermatogonia and an increase in DNA double-strand breaks. Exposure to 150 and 0.15 μg/L of ZEA has been shown to reduce sperm concentration and increase the occurrence of morphologically abnormal spermatozoa, while higher doses can disrupt spermatogenesis altogether. Reportedly, ZEA can influence reproductive ability by affecting the synthesis and secretion of hormones such as testosterone, estradiol, and progesterone in both males and females. Studies have also shown that mice experience decreased testosterone and estrogen levels following oral administration of ZEA for 14 days. Feeding pregnant rats a ZEA-contaminated diet resulted in a decrease in estradiol levels in the mothers and a reduction in serum testosterone concentrations in the first generation of adult male offspring.

The mammalian target of rapamycin (mTOR) is an atypical serine-threonine protein kinase that exists in two distinct complexes. mTOR complex 1 (mTORC1) serves as a primary growth regulator, sensing and integrating various nutrient and environmental cues, including growth factors, energy levels, cellular stress, and amino acids. It combines these signals to promote cell growth by phosphorylating substrates that enhance anabolism or limit catabolism. Given that mTOR activity is sensitive to cellular energy and nutritional status, it can be regulated by AMPK. Studies have reported that AMPK can promote autophagy by inhibiting mTOR. Previous research has indicated that ZEA increases the level of autophagy in TM4 cells. However, whether this promotion occurs via the AMPK/mTOR/P70S6K signaling pathway requires further investigation. The mTOR/P70S6K signaling pathway also plays a crucial role in regulating testicular development and spermatogenesis. Therefore, studying the AMPK signaling pathway is essential for a deeper understanding of the toxicological mechanisms of ZEA in reproduction.

AMPK is a highly conserved cellular energy and metabolic regulator with critical roles in regulating energy metabolism in almost all cells under both physiological and pathological conditions. It is not only activated by AMP-induced phosphorylation but is also phosphorylated and activated by the tumor suppressor LKB1 or the Ca2+/CaMKKβ complex. The activation of AMPK by CaMKKβ occurs in response to an increase in intracellular Ca2+ concentration.

Previous findings from our laboratory demonstrated that ZEA induces endoplasmic reticulum (ER) stress and elevates Ca2+ concentration in Sertoli cells (SCs). Consequently, the present study aims to investigate whether intracellular calcium ions are released from the ER and subsequently induce autophagy through CaMKKβ-regulated activation of AMPK and the resulting inhibition of mTOR activity. Collectively, the outcomes of this study are expected to provide a theoretical foundation for the prevention and treatment of ZEA poisoning.

2. Materials and methods

2.1. Reagents

Zearalenone (ZEA) was obtained from Sigma-Aldrich (USA). DMEM/F-12 medium and fetal bovine serum (FBS) were purchased from GIBCO BRL (USA). 4,6-Diamidino-2-phenylindole (DAPI), 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), BCA protein assay kit, and Fluo-3-pentaacetoxymethyl ester (Fluo-3AM) were acquired from Beyotime Institute of Biotechnology (Shanghai, China). Compound C was purchased from Abcam (USA). AMPK siRNA was obtained from Santa Cruz Biotechnology (USA). Rabbit monoclonal antibodies against GAPDH, P62, AMPK, p-AMPK, mTOR, p-mTOR, P70S6K, and p-P70S6K were purchased from Cell Signaling Technology (USA). LC3 antibody was obtained from Sigma-Aldrich (USA).

2.2. Cell culture

TM4 cells (mouse Sertoli cell line) were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% penicillin, and 1 mM glutamine. TM4 cells were maintained at 37℃ in a humidified atmosphere containing 5% CO2.

2.3. Cell proliferation assay

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). Cells were seeded at a density of 1 × 104 cells per well in a 96-well plate. Control wells, blank wells, and experimental wells (containing Compound C at 5 μmol/L or AICAR at 50 μmol/L) were prepared separately, with six replicates for each group. CCK-8 solution (10 μM) was added to each well, and the culture plate was incubated for 2-4 hours. Subsequently, the absorbance was measured at 450 nm.

2.4. Measurement of intracellular Ca2+

Fluo-3AM, a calcium indicator, exhibits increased fluorescence upon binding to Ca2+, a property utilized to detect changes in intracellular Ca2+ levels. Following the designated treatments outlined in the study design, cells were incubated with Fluo-3AM at 37℃ in darkness for 30 minutes. Subsequently, the cells were analyzed using a flow cytometer with an excitation wavelength of 488 nm to quantify intracellular calcium levels.

2.5. Western blotting analysis

Total cellular proteins were extracted using RIPA lysis buffer, and protein concentration was determined using the BCA Protein Assay Kit. The extracted proteins were mixed with loading buffer and boiled for 10 minutes. Protein samples (20 μg per lane) were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels and then transferred to nitrocellulose membranes using electrophoresis and Trans-Blot Turbo systems (BIO-RAD). The membranes were blocked with 5% non-fat milk for 2 hours at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4℃, washed five times with TBST, and then incubated with secondary antibodies for 2 hours at room temperature. After five washes with TBST, protein signals were detected using an ECL detection system.

2.6. Transfection with small-interfering RNA (siRNA)

TM4 cells in the logarithmic growth phase were cultured in 60-mm dishes until they reached 60% confluency. Transfection with AMPK siRNA or a scrambled control sequence was performed using Lipo 3000 and Opti-MEM, following the manufacturer’s instructions. After a 15-minute incubation at room temperature, the resulting complexes were added dropwise onto the cells in replaced culture medium. The cells were maintained in a 37°C incubator for 6 hours, after which the medium was replaced with complete medium until analysis. Forty-eight hours post-transfection, the cells were harvested for protein expression analysis of AMPK.

2.7. The Immunofluorescence of TM4 cells

Cells were seeded on sterile glass coverslips at a density of 4 × 104 cells per coverslip. Following exposure to different treatment groups for 24 hours, the cells were fixed with 4% paraformaldehyde at 4℃ for 30 minutes, washed three times with PBS, and then permeabilized with 0.5% Triton X-100 at room temperature for 20 minutes. After three washes with PBS, the cells were incubated with 5% BSA blocking solution at room temperature for 30 minutes.

2.8. Statistical analysis

All experimental results were subjected to statistical analysis using one-way ANOVA with SPSS 22.0 statistical software. Multiple comparisons were performed using the LSD method. Data are presented as mean ± standard deviation (SD). A probability (P) value of less than 0.05 was considered statistically significant.

3. Results

3.1. Role of AMPK/mTOR/P70S6K signaling pathway in ZEA-induced autophagy in TM4 cells

The AMPK/mTOR/P70S6K pathway is a key signaling cascade involved in the regulation of autophagy. Our previous findings indicated that ZEA induces autophagy in TM4 cells. Therefore, the present study investigated the role of this signaling pathway in ZEA-induced autophagy in these cells. A concentration of 5 μmol/L of Compound C was selected for these experiments. Western blot analysis following pretreatment with this AMPK inhibitor showed a reduction in both total AMPK protein and phosphorylated AMPK. Compared to the group treated with ZEA alone, the combined treatment of Compound C and ZEA resulted in significantly decreased expression levels of p-AMPK/AMPK, while the expression of p-mTOR/mTOR and p-P70S6K/p70S6K was significantly increased. Importantly, compared to the ZEA-only treatment, the combined treatment with Compound C and ZEA led to a marked decrease in the expression of LC3II/LC3I, whereas the expression of Beclin1 and P62 did not show significant differences. Consistently, immunofluorescence analysis of TM4 cells pretreated with Compound C demonstrated a notable reduction in LC3 puncta and a decrease in the overall level of LC3, which aligns with the Western blot results.

Subsequently, the AMPK activator AICAR was added to TM4 cells for further investigation. A concentration of 50 μmol/L of AICAR did not exhibit any toxic effects on the cells and was therefore chosen for subsequent experiments. Moreover, AICAR did not affect the total expression of AMPK but enhanced the activity of p-AMPK. The combined treatment of AICAR and ZEA significantly increased the levels of p-AMPK/AMPK compared to the ZEA-only group; similarly, LC3-II/LC3-I and Beclin-1 levels were significantly elevated. Immunofluorescence analysis revealed a higher number of LC3 puncta in AICAR-pretreated TM4 cells compared to cells incubated with ZEA alone or without ZEA, which was consistent with the Western blot analysis.

3.2. Role of AMPK in modulating AMPK/mTOR/P70S6K signaling and ZEA-induced autophagy

To further elucidate the role of AMPK in ZEA-induced autophagy, siRNA was employed to inhibit AMPK expression. Transfection with 33 nmol/L AMPK siRNA resulted in a significant reduction in AMPK expression. Furthermore, the expression of the downstream proteins, p-P70S6K/p70S6K, was significantly increased compared to cells transfected with non-targeting control siRNA. Importantly, the expression levels of LC3II/LC3I were significantly reduced in TM4 cells transfected with AMPK siRNA. These findings indicate that AMPK acts as a key upstream molecule in the mTOR/P70S6K signaling pathway and plays a crucial role in ZEA-induced autophagy in TM4 cells.

3.3. ZEA-induced ER stress activates AMPK via Ca2+/CaMKKβ leading to autophagy induction

When cells were treated with varying concentrations of ZEA for 24 hours, flow cytometry analysis revealed changes in Ca2+ concentration. The intracellular calcium level increased with increasing ZEA concentrations, indicating that ZEA exposure elevates intracellular calcium ion concentration.

Pretreatment with BAPTA-AM, a specific calcium chelator, resulted in a marked decrease in Ca2+ concentration compared to the ZEA-only treated group, as detected by flow cytometry. To further investigate whether intracellular calcium ions originated from the endoplasmic reticulum (ER), TM4 cells were treated with 2-APB, an inhibitor of ER calcium ion release channels. This treatment also led to an apparent decrease in intracellular calcium levels, suggesting the ER as a source of the released calcium ions.

Elevated cytoplasmic Ca2+ concentrations have been reported to stimulate autophagy through Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ) and subsequent AMPK activation. In this study, treatment of TM4 cells with different ZEA concentrations for 24 hours increased the expression of CaMKKβ. Notably, the CaMKKβ inhibitor STO-609 (STO) reduced the ZEA-induced upregulation of both CaMKKβ and p-AMPK, indicating that CaMKKβ acts upstream of AMPK and activates it. Furthermore, the ER calcium ion release channel inhibitor 2-APB attenuated the expression of Bip and CHOP, markers of ER stress. Moreover, compared to the ZEA-only treatment, the combined treatment of 2-APB and ZEA resulted in a marked decrease in the expression of LC3II/LC3I, an autophagy marker. Analysis of p-CaMKKβ, p-AMPK/AMPK, p-mTOR/mTOR, and p-P70S6K/P70S6K expression levels revealed that the combined treatment of 2-APB and ZEA significantly decreased the expression of p-CaMKKβ and p-AMPK/AMPK while increasing the expression of p-mTOR and p-P70S6K compared to the ZEA-only treatment. These findings suggest that ZEA-induced ER stress leads to increased calcium release from the ER lumen, followed by CaMKKβ-induced activation of the AMPK/mTOR/P70S6K signaling pathway, ultimately resulting in autophagy activation.

3.4. ZEA induced ER stress via reactive oxygen species (ROS) and activated AMPK/mTOR/P70S6K signaling pathway to regulate autophagy

To further investigate the potential link between ZEA-induced autophagy and the production of reactive oxygen species (ROS), the antioxidant N-acetyl-L-cysteine (NAC) was used. Compared to the ZEA treatment group, the fluorescence puncta of LC3 were significantly decreased in the group treated with both NAC and ZEA. Western blot analysis also showed that NAC pretreatment reduced the expression of LC3, consistent with the immunofluorescence results. Moreover, this study found that NAC attenuated the ZEA-induced upregulation of p-AMPK expression while enhancing the expression of p-mTOR and p-P70S6K expression. These findings demonstrate that ROS regulates AMPK-mediated ZEA-induced autophagy in TM4 cells.

4. Discussion

Accumulating evidence suggests that autophagy plays a regulatory role in spermatogenesis damage. Previous research indicated that ZEA is cytotoxic and induces autophagy in Sertoli cells (SCs). The present study elucidated the molecular mechanisms underlying ZEA-induced autophagy, concluding that calcium ions released due to ROS-mediated ER stress activate CaMKKβ, which in turn activates AMPK, a key regulator of autophagy.

The AMPK/mTOR/P70S6K pathway is a central signaling cascade governing autophagy. AMPK acts as a cellular energy sensor, regulating cellular energy metabolism and maintaining intracellular homeostasis. During cellular stress, an energy crisis occurs, leading to decreased ATP concentration, increased AMP concentration, an elevated intracellular AMP/ATP ratio, and subsequent AMPK activation. AMPK can also be activated by upstream CaMKKβ kinases. Previous studies have shown that AMPK phosphorylation positively regulates autophagy through various downstream signaling molecules. Notably, mTOR, a downstream target of AMPK, inhibits autophagy and promotes cell growth and proliferation by phosphorylating the 40S ribosomal protein S6 kinase (P70S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1).

To further clarify the role of AMPK in autophagy regulation, AMPK inhibitors, an activator, and siRNA were employed, and the AMPK signaling pathway along with the expression of autophagy-related proteins were assessed. Pretreatment with Compound C or AMPKα siRNA effectively blocked ZEA-induced AMPK activation, thereby inhibiting autophagy in TM4 cells. Conversely, the AMPK activator AICAR significantly enhanced ZEA-induced autophagy in TM4 cells via the AMPK/mTOR/P70S6K pathway. These results demonstrate that ZEA regulates autophagy through the AMPK signaling pathway, providing new insights into the mechanisms of testicular dysfunction caused by ZEA exposure.

The endoplasmic reticulum (ER) serves as a reservoir of calcium ions. ER stress, characterized by misfolding and aggregation of unfolded proteins within the ER lumen, disrupts calcium ion homeostasis. Furthermore, CaMKKβ activation is primarily dependent on conformational changes induced by the binding of Ca2+ and calmodulin. Therefore, the level of free Ca2+ in the cytoplasm is crucial for CaMKKβ activation. Previous findings confirmed that ZEA induces ER stress in SCs, leading to an increase in Ca2+ concentration. Moreover, the intracellular calcium inhibitor BAPTA-AM reduces calcium levels. The subsequent addition of the ER calcium channel release inhibitor 2-APB further decreased calcium levels, indicating the ER as the source of intracellular calcium ion release. Studies have suggested that increased calcium ion concentration activates CaMKKβ and AMPK to regulate autophagy. The current study demonstrated that the addition of STO609 inhibits AMPK expression, confirming that CaMKKβ can activate AMPK. Additionally, the activation of CaMKKβ by increased calcium ion concentration was validated. The results of this study demonstrate that CaMKKβ is an upstream regulator of AMPK during ZEA exposure, which induces autophagy. Thus, it can be concluded that increased calcium ion concentration activates CaMKKβ.

Recent research has identified ROS as an upstream signaling molecule that regulates autophagy initiation, and previous studies have shown that abundant ROS can activate AMPK. Furthermore, mycotoxins induce oxidative stress and generate ROS. Our previous studies also confirmed that ZEA produces ROS and causes damage to testicular support cells. Several studies have indicated that ROS mediates ER stress, playing a regulatory role in various drug-induced ER stress processes. Moreover, ROS can oxidize and inactivate key thiols in the ryanodine receptor, thereby enhancing Ca2+ release from the sarcoplasmic reticulum. ROS production in a mouse hypoxia-ischemia model causes oxidative modification of ER proteins. MnTMPyP, a mimic of the ROS inhibitor MnSOD, inhibits ROS increase in the ER. N-acetylcysteine is frequently used as an antioxidant to scavenge ROS and block ROS production. The current study suggested that ZEA-induced ER stress was alleviated, and Ca2+ concentration was reduced by NAC.

Furthermore, ROS acts as an upstream signaling molecule regulating autophagy initiation. Studies have shown that high levels of ROS activate AMPK, and Bri induces autophagy by activating ROS-regulated AMPK in liver cancer cells. In alcoholic liver disease studies, ethanol modeling induces oxidative damage, producing ROS, which in turn induces autophagy by activating AMPK. Therefore, these results confirmed that ZEA-induced autophagy in TM4 cells via the AMPK/mTOR/P70S6K signaling pathway was inhibited by NAC administration. Taken together, ROS is an upstream molecule of the AMPK/mTOR/P70S6K signaling pathway and regulates autophagy mTOR inhibitor.

5. Conclusions

In summary, this study indicates that ZEA induces ER stress through ROS production, leading to increased calcium release from the ER and subsequent activation of the CaMKKβ/AMPK signaling pathway, ultimately regulating autophagy.