The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Abstract

Oxysterols, such as 7beta-hydroxy-cholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca(2+)) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca(2+) changes on apoptosis induced by 7beta-OH and beta-epoxide. Ca(2+) responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura-2. Over 15-min exposure of differentiated U937 cells to 30 muM of 7beta-OH induced a slow but significant rise in fura-2 ratio. The Ca(2+) channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca(2+). Moreover, dihydropyridine (DHP) binding sites identified with BODIPY-FLX-DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca(2+) occurred through L-type channels. However, following long-term incubation with 7beta-OH, elevated levels of cytoplasmic Ca(2+) were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca(2+) may be an initial trigger of 7beta-OH-induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca(2+) on apoptotic cell death appears to be less significant. In contrast, Ca(2+) did not appear to be involved in beta-epoxide-induced apoptosis.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

“…However, the fact that the effect of oxLDL was mimicked by exogenous S1P and inhibited by an SK inhibitor suggests that S1P plays a major role. In addition, a recent study in U937 macrophages found that increased intracellular Ca 2+ in response to 7 ␤ -hydroxycholesterol was mediated by infl ux of extracellular Ca 2+ and was nonoscillatory ( 57 ). Both of these features are different from our fi ndings with oxLDL-induced Ca 2+ signaling in BMDM.…”

Macrophage oxysterols and their binding proteins

“…Different oxysterols have distinct impacts on membrane lipid packing [20,21] and thereby interfere with the cholesterol-sphingolipid-enriched raft domains with key roles in a number of signal transduction events [22]. In many cases, increases in cytoplasmic (Ca 2þ ) are the earliest responses to oxysterol or oxidized LDL [23,24], followed by inhibition of prosurvival phospoinositide 3-kinase (PI3K)/3-phosphoinositide dependent protein kinase-1(PDK-1)/Akt signaling [25,26], activation of the Ca 2þ dependent phosphatase calcineurin [27], induction of distinct proapoptotic routes involving the Bad, Bim, or Bax proteins [25,[27][28][29] and suppression of the antiapoptotic Bcl-2/Bcl-xL proteins [25,[29][30][31]. Much of the cytotoxicity attributable to oxysterols is due to their ability to induce apoptosis.…”

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13

“…We therefore investigated the possible relationships between the NGR-peptide-1 lethal effects, Ca 2+ release and ROS production in U937 cells. Firstly, we analyzed the ability of two Ca 2+ chelators (the cell-impermeant compound BAPTA and the cell-permeant compound BAPTA-AM) and nifedipine (known to block L-type Ca 2+ channels in U937 cells [ 50 ]) to modulate NGR-peptide-1-induced cell death (as determined by annexin-V-FITC/PI staining). In absence of NGR-peptide-1, these inhibitors did not alter surface CD13 levels.…”