Mechanism of oxLDL causing CHOP activation during ER stress and its inhibition by macrophage antioxidant 7,8-dihydroneopterin in U937 cells
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Endoplasmic Reticulum stress (ER) and oxidative stress are two related processes which can cause the death of macrophages within the artery wall. Atherosclerosis is a chronic inflammatory disease caused by the abnormal deposition of cholesterol ester filled macrophages within the inner arterial wall so limiting flow of oxygenated blood to various organs of the body. Scavenger receptors such as CD36 are responsible for the absorption of heavily oxidized low density lipoprotein (oxLDL) forming atherosclerotic plaque in the artery wall. The macrophage antioxidant 7, 8-dihydroneopterin downregulate macrophage CD36 so preventing cholesterol esters accumulations in the cells. The ER-stress triggered by free unesterified cholesterol accumulation causes perturbations in ER function resulting in incorrectly folded proteins. ER stress causes activation of three ER proteins resulting in signaling pathways that lower ER stress as a part of Unfolded Protein Response (UPR). However, unresolved ER-stress leads to the activation of the CHOP (C/EBP-homologous protein) protease which causes mitochondrial-mediated cell death. The research investigated the effect of oxLDL causing CHOP activation in U937 cell line and its inhibition by 7, 8-dihydroneopterin.
Thapsigargin (TG), an ER calcium agonist, was found to be a suitable positive control of ER stress-mediated cell death in U937 cells. TG was very cytotoxic causing a 70% loss on cell viability when treated with at only 100 nM. The mitochondrial membrane potential surprisingly increased with TG treatment but at the same time there was a large loss of intact cells which may have masked the loss of mitochondrial potential.
Western blot analysis showed 50 nM TG caused maximum expression of ER-stress marker CHOP in U937 cells. The expression increased with increasing concentrations of TG suggesting the potential of TG in triggering CHOP activation through the unresolved ER-stress-UPR pathway. Cu-oxLDL caused CHOP expression in U937 cells with increasing concentrations with activation occurring within 3 hours with maximum levels seen by 24 hours.
The addition of the macrophage generated antioxidant 7, 8-dihydroneopterin reduced the CHOP expression in U937 cells. Treatment with 200 μM 7, 8-dihydroneopterin reduced the levels of CHOP to 57.67% of control.
ACAT inhibitor (Sandoz 580-35) was expected to trigger ER-stress through the buildup of free cholesterol. Instead the inhibitor caused an increase in cell survival when the U937 cells treated with a sub-lethal dose of oxLDL. The inhibitor prevented cell death at lower concentrations of 10 μg/mL. However, it was observed that the ACAT inhibitor is lethal to cells when given at 25 μg/mL and above concentrations in the absence of oxLDL.
The results confirm that TG and oxLDL induced cell death in U937 cells through ER-stress pathway by CHOP activation. The results showed the potential of macrophage antioxidant 7, 8-dihydroneopterin in downregulating CHOP expression suggesting the suppression of CHOP-induced ER-stress in the cells. The research also demonstrated that ACAT inhibitor at lower concentrations can increase the cell survival rates during oxLDL induced cell death in U937 cells.