Parkin Modulates ERRα/eNOS Signaling Pathway in Endothelial Cells

Abstract

Background/Aims: Although a number of reports documented the important role of parkin in mitophagy, emerging evidence also indicated additional functions of parkin besides mitophagy. The present study was undertaken to investigate the role of parkin in the regulation of ERRα/eNOS pathway in endothelial cells (ECs). Methods: Mouse aortic endothelial cells (MAECs) and cardiac muscle HL-1 cells were transfected with parkin plasmid or siRNA. ERRα inhibitor XCT-790, autophagy inhibitor 3-MA and Bafilomycin A1, and caspase inhibitor Z-VAD-FMK were used to block autophagy or apoptosis. Western blotting was performed to examine the protein levels. Flow cytometry was applied to determine the cell apoptosis and ROS production. Mitochondrial membrane potential was measured using JC-1 and TMRM. Immunoprecipitation was performed to confirm the parkin effect on ERRα ubiquitination. Results: Overexpression of parkin resulted in a significant reduction of total-eNOS and p-eNOS in parallel with the downregulation of ERRα (a regulator of eNOS) protein and the enhancement of ERRα ubiquitination. To test the role of ERRα in regulating eNOS in this experimental setting, we treated ECs with ERRα inhibitor and found a decrement of total-eNOS and p-eNOS. On the contrary, overexpression of ERRα increased the levels of total-eNOS and p-eNOS. Meanwhile, parkin overexpression induced mitochondrial dysfunction and cell apoptosis in both ECs and HL-1 cells. Finally, we confirmed that the parkin effect on the regulation of eNOS was independent of the autophagy and apoptosis. Conclusion: These findings suggested that parkin overexpression downregulated eNOS possibly through the ubiquitination of ERRα in endothelial cells.