Apoptosis signal-regulating kinase 1 promotes Ochratoxin A-induced renal cytotoxicity

Abstract

Oxidative stress and apoptosis are involved in Ochratoxin A (OTA)-induced renal cytotoxicity. Apoptosis signal-regulating kinase 1 (ASK1) is a Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK, MAP3K) family member that plays an important role in oxidative stress-induced cell apoptosis. In this study, we performed RNA interference of ASK1 in HEK293 cells and employed an iTRAQ-based quantitative proteomics approach to globally investigate the regulatory mechanism of ASK1 in OTA-induced renal cytotoxicity. Our results showed that ASK1 knockdown alleviated OTA-induced ROS generation and Δψm loss and thus desensitized the cells to OTA-induced apoptosis. We identified 33 and 24 differentially expressed proteins upon OTA treatment in scrambled and ASK1 knockdown cells, respectively. Pathway classification and analysis revealed that ASK1 participated in OTA-induced inhibition of mRNA splicing, nucleotide metabolism, the cell cycle, DNA repair, and the activation of lipid metabolism. We concluded that ASK1 plays an essential role in promoting OTA-induced renal cytotoxicity.

Figure 1. ASK1 was activated by OTA.

(A): HEK293 cells were treated with 20 μM OTA for the indicated times, and western blot was used to detect the phosphorylation (Thr838) of ASK1. (B): Relative expression of p-ASK1 after 20 μM OTA treatment was shown as total gray value using BandScan software. The p-ASK1 expression in 0 h was normalized to 1. Asterisk (*) indicates significant differences (p < 0.05). Gels were run under the same experimental conditions while images of western blots displayed in cropped format.

Figure 2. Confirmation of ASK1 knockdown efficiency.

(A): Western Blot validation of ASK1 expression in knockdown cells versus scrambled cells. (B): Relative expression of ASK1 in knockdown cells versus scrambled cells was shown as total gray value using BandScan software. The ASK1 expression of scrambled cells was normalized to 1. Asterisk (*) indicate significant differences (p < 0.05). Gels were run under the same experimental conditions while images of western blots displayed in cropped format.

Figure 3. Cell viability of HEK293 cells in response to OTA treatment.

Scrambled cells and ASK1 knockdown cells were exposed to increasing concentration of OTA for 24 h and determined by WST-8 staining. Different lowercase letters indicate significant differences (p < 0.05).

Figure 4. ROS generation of HEK293 cells in response to OTA treatment.

Scrambled cells and ASK1 knockdown cells were exposed to 20 μM of OTA for 1 h or 24 h. Cells were stained by DCFH-DA fluorescent probe and determined by flow cytometry. Asterisk (*) indicate significant differences (p < 0.05).

Figure 5. Mitochondrial membrane potential (Δψm) of HEK293 cells in response to OTA treatment.

Scrambled cells and ASK1 knockdown cells were exposed to 20 μM of OTA for 1 h or 24 h. Cells were stained by JC-1 and fluorescence was determined by microplate reader. Δψm was expressed as the ratio of red fluorescence over green fluorescence. Asterisk (*) indicate significant differences (p < 0.05).

Figure 6. Schematic representation of the experimental design for iTRAQ labeling.

Figure 7. An overview of differentially expressed proteome.

(A): Venn diagram depicted the overlap of proteins identified by iTRAQ measurements among three biological replicates. (B): Whetton's plot. Log ratios for all proteins in the OTA treatment versus control were plotted against the number of peptides.

Figure 8. Proteins classified into distinct pathway categories using DAVID functional annotation and UniProt annotation.

For each Venn diagram, Left circle indicates differentially expressed proteins in scrambled cell group (OTA treatment versus control) and right circle indicates those in ASK1 knock-down cell group (OTA treatment versus control). The overlapping section indicates proteins both appeared in the two cell groups. Protein names in red represent up-regulated proteins and those in blue represent down-regulated proteins.

Figure 9. Validation of iTRAQ data by Western Blot.

Four proteins were selected to validate the alteration trend using Western Blot. Gels were run under the same experimental conditions while images of western blots displayed in cropped format. KRT18 was up-regulated in ASK1 knockdown cell group and PKM was up-regulated in scrambled cell group compared with control (no drug treatment). CDK1 and DHRS2 were both down-regulated in scrambled cell group as well as ASK1 knockdown cell group compared to control. Actin was used as a loading control.

Figure 10. A hypothetical model of ASK1 mediating OTA-induced cytotoxicity.

(↑) indicates proteins that were activated or up-regulated and (↓) indicates proteins that were inhibited or down-regulated.