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Ochratoxin A induces ERK1/2 phosphorylation-dependent apoptosis through NF-κB/ERK axis in human proximal tubule
Elif Darbuka1, Can Gürkaşlar1, Ibrahim Yaman2
1Bogazici University, Department of Molecular Biology and Genetics, Molecular Toxicology and Cancer Research Laboratory, Bebek, Istanbul, 34342, Turkey.
Abstract:
Ochratoxin A (OTA) is a food contaminant mycotoxin with hazardous effects on human and animal health, primarily affecting the kidneys. OTA's mode of action is not well understood. OTA activates both MAPK/ERK and PI3K/Akt signaling pathways, which play role in apoptosis and cell survival, respectively. OTA is also known to induce toxicity by activating the NF-κB pathway in immune cells. However, its role in determining the cell fate upon OTA exposure in a human kidney cell line (HK-2) has not been fully explored. We made use of pharmacological inhibition of NF-κB to define its role in viability of OTA-treated HK-2 cells. We show that OTA-induced p65 NF-κB subunit translocation into the nucleus in a time-dependent manner using both Western blotting and immunofluorescence (IF). We also document the DNA-binding and reporter gene expression activities of NF-κB by electrophoretic mobility shift (EMSA) and luciferase reporter assays, respectively. Our results indicate that, following 6 h of exposure, OTA fully activates NF-κB pathway and its downstream effectors in HK-2 cells. In addition, Bay11-7085 treatment causes attenuation of the relative levels of OTA-mediated ERK1/2 phosphorylation, suggesting a cross-talk between NF-κB and the MAPK/ERK pathway. Critically, co-treatment of HK-2 cells with OTA and Bay11-7085 leads to the inhibition of OTA-induced apoptosis in a time-dependent manner. Our results support a robust association between NF-κB and the MAPK/ERK pathways in the modulation of apoptotic effects of OTA in HK-2 cells.
Insights
Ochratoxin A (OTA) triggers kidney cell death by activating NF-κB and MAPK/ERK pathways. Inhibiting NF-κB with Bay11-7085 reduces OTA-induced apoptosis, revealing a key interaction in kidney toxicity.
Area of Science:
- Toxicology
- Molecular Biology
- Cell Biology
Background:
- Ochratoxin A (OTA) is a prevalent food contaminant mycotoxin with significant nephrotoxic effects.
- The precise molecular mechanisms underlying OTA-induced kidney toxicity, particularly its impact on cell fate, remain incompletely understood.
- While OTA is known to activate MAPK/ERK, PI3K/Akt, and NF-κB signaling pathways, its specific role in human kidney cells (HK-2) requires further elucidation.
Purpose of the Study:
- To investigate the role of the Nuclear Factor-kappa B (NF-κB) pathway in Ochratoxin A (OTA)-induced apoptosis in human kidney HK-2 cells.
- To explore the potential cross-talk between the NF-κB and MAPK/ERK signaling pathways in response to OTA exposure.
- To determine if pharmacological inhibition of NF-κB can mitigate OTA-induced kidney cell death.
Main Methods:
- Utilized Western blotting and immunofluorescence (IF) to assess NF-κB p65 subunit translocation.
- Employed electrophoretic mobility shift assays (EMSA) and luciferase reporter assays to evaluate NF-κB DNA-binding and transcriptional activity.
- Administered pharmacological inhibitor Bay11-7085 to block NF-κB activation and assessed its effects on OTA-induced apoptosis and ERK1/2 phosphorylation.
Main Results:
- OTA exposure led to time-dependent nuclear translocation of the p65 NF-κB subunit in HK-2 cells.
- NF-κB pathway was fully activated by OTA within 6 hours, as evidenced by DNA-binding and reporter gene activity.
- Inhibition of NF-κB by Bay11-7085 attenuated OTA-induced ERK1/2 phosphorylation, indicating pathway cross-talk.
- Co-treatment with OTA and Bay11-7085 significantly inhibited OTA-induced apoptosis in a time-dependent manner.
Conclusions:
- The NF-κB pathway is a critical mediator of Ochratoxin A-induced apoptosis in human kidney HK-2 cells.
- A significant cross-talk exists between the NF-κB and MAPK/ERK signaling pathways in modulating OTA toxicity.
- Pharmacological inhibition of NF-κB presents a potential therapeutic strategy to counteract OTA-induced nephrotoxicity.
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