The uptake mechanism and intracellular fate of Paraoxonase-1 in endothelial cells.

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated lactonase that plays a significant role in the anti-atherosclerotic activity of HDL. However, several studies have shown that PON1 localizes in cells, where it operates independently of HDL. Previously, we showed that PON1 localizes in endothelial cells (ECs), and impairs vasodilation mediated by the endothelium-derived hyperpolarizing factor (EDHF) 5,6-δ-DHTL. However, the internalization pathway of PON1 into ECs, and the intracellular fate of PON1 are unknown. Therefore, the present study aimed to elucidate the uptake mechanism, intracellular trafficking and the function of PON1 in ECs. ‏We conducted a series of inhibition experiments of fluorescently labeled recombinant PON1 (rePON1) in ECs, followed by FACS analyses. We found that rePON1 binds the EC membrane via specific binding sites located in lipid-rafts/caveolae microdomains that are shared with HDL, and internalized through dynamin-dependent endocytosis. Qualitative assessments of the intracellular trafficking of rePON1, using confocal z-stack images, showed colocalization of the labeled rePON1 with early and late endosome/lysosome markers. Accordingly, a "pulse-chase" incubation of rePON1, followed by lactonase activity measurement in EC lysate, revealed that rePON1 retains its lactonase activity after binding to the cells. However, this activity decreases over time. Finally, induction of endothelial dysfunction with high glucose, angiotensin II, or palmitic acid increased rePON1 uptake by ECs. In conclusion, these results indicate that free PON1 interacts with ECs via binding sites located in lipid-rafts/caveolae, where it is enzymatically active and regulates endothelial functions. However, once internalized, PON1 is degraded. Additionally, alteration in endothelial function affects PON1 uptake by ECs.