Modification of protein disulfide isomerase by lipid oxidation products contributes to oxidized low density lipoprotein-mediated apoptosis

Carole Muller, Jan Bandemer, Cecile Vindis, Robert Salvayre, Anne Negre-Salvayre. Team 10, INSERM U858 and University Paul Sabatier, Toulouse, CEDEX 4, 31432, France.

Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response (UPR), which tends to restore ER homeostasis, but switches to apoptosis when ER stress is prolonged. ER stress is induced by oxidized LDL (oxLDLs) and lipid oxidation products, and contributes in part to oxLDL-mediated apoptosis of vascular endothelial cells. Besides, ER-associated chaperones are antiapoptotic, such as the protein disulphide isomerase (PDI), which participates to the maturation of newly synthesized proteins by correcting disulfide bonds. The aim of this work was to investigate the implication of PDI in oxLDL-induced apoptosis.

Toxic oxLDL concentrations (200 µg/ml) and lipid oxidation products 7-ketocholesterol (10 µmol/L) and 4-hydroxynonenal (20 µmol/L), induced ER stress in human endothelial cells HMEC-1, as assessed by the activation of ER stress sensors (phosphorylation of Ire1alpha and PERK, nuclear translocation of ATF6) and of their subsequent targets (eIF2alpha phosphorylation), as well as increased expression of XBP1/spliced XBP1, of the prapoptotic factor CHOP and of KDEL chaperones (Sanson et al. 2009). OxLDLs (200 µg/mL) inhibited PDI enzyme activity (50 % PDI inhibition after 24 h incubation with 200 µg/mL, mean ± SEM of 4 separate experiments), without modifying PDI protein expression. OxLDLs (200 µg/mL) were toxic for HMEC-1 (25 % cell viability after 24 h incubation with 200 µg/mL oxLDL, mean± SEM of 4 separate experiments) To check whether PDI is involved in oxLDL-induced cell death, PDI was inhibited by bacitracine (100 nmol/L) or by transfection with a vector coding for a mutant inactive form. PDI inhibition resulted in an increased expression of ER stress markers such as spliced XBP1 and CHOP, and in increased apoptosis induced by non-toxic oxLDL concentrations (45 % cell viability after 24h, in cell incubated with bacitracine + 100 µg/mL oxLDL, vs 80% in cells incubated with oxLDL alone) (these results are mean ± SEM of 4 separate experiments). The inhibitory effect of oxLDLs could result from the formation of 4-hydroxynonenal (4-HNE)-adducts on PDI. The antioxidant and carbonyl scavenger N-acetyl cysteine (NAC) (10 mol/L) inhibited the formation of 4-HNE adducts on PDI, restored its basal enzymatic activity and inhibited oxLDL-induced ER stress and apoptosis (80% cell viability evaluated by the MTT test in cells incubated with 200 µg/mL oxLDL+NAC, vs 25 % cell viability in cells incubated with oxLDLs alone).

In conclusion, PDI modification by 4-HNE, impairs its protective antiapoptotic function and aggravates ER stress and apoptosis induced by oxLDLs. NAC neutralizes the modification of PDI by 4-HNE and inhibits ER stress induction and subsequent apoptosis.

Sanson M, et al. (2009) Circ Res. 104, 328-336