NAD(P)H Oxidase Nox-4 Mediates 7-Ketocholesterol-Induced Endoplasmic Reticulum Stress and Apoptosis in Human Aortic Smooth Muscle Cells

Abstract

The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca 2؉ oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH 2 -terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.Atherosclerosis is a slow degenerative process and is the underlying cause of heart attacks, strokes, and peripheral artery diseases in humans. This complex disorder is characterized by a remodeling of the arterial wall, leading to the formation of an atherosclerotic plaque. Plaque formation is induced by the accumulation, at the subendothelial level, of oxidized low-density lipoproteins (LDLs) and subsequently of some of their lipid constituents (oxysterols, oxidized fatty acids, aldehydes, and lysophospholipids) and fibrous elements.To date, a number of studies have shown that oxysterols constitute an important family of oxygenated derivatives of cholesterol that exert potent biological effects in the pathogenesis of atherosclerosis (for a review, see references 6 and 9). Among the oxysterols that have been identified, those oxidized at the C7 position, such as 7-ketocholesterol (7-Kchol), are the ones most frequently detected at high levels in atherosclerotic plaques (9) and in the plasma of patients with high cardiovascular risk factors (55). 7-Kchol exerts deleterious effects on vascular smooth muscle cells (SMCs), including the stimulation of reactive oxygen species (ROS) production (28) and the induction of apoptosis (30,34,42), two major events involved in atherogenesis. The oxidation of macromolecules (proteins, lipids, and DNA) and apoptosis induce the progression of atherosclerosis. Thus, the death of vascular SMCs and monocyte-derived foam cells has been shown to modulate the cellularity of...

7‐Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7‐ketocholesterol–induced apoptosis: Analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation

“…Further, because free cholesterol activates apoptosis through the endoplasmic reticulum pathway (57), the intracellular accumulation of free cholesterol triggered by 7KC can stimulate some components of the endoplasmic reticulum, which in turn can induce 7KC-induced apoptosis. This hypothesis is supported by the expression of the cell death effector CHOP and GRP78/ bip chaperone, which are hallmarks of the unfolded protein response (58), and by the synthesis of similar cytoplasmic structures occurring under treatment with 7KC and free cholesterol (22,23,57,59). At the cytoplasmic level, the presence of myelin figures observed during 7KC-induced apoptosis (22,23) evokes phospholipid whorls formed during cell death of murine macrophages induced by free cholesterol (54).…”

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

“…Pedruzzi et al [16] reported that Nox4 activity located in the endoplasmic reticulum was concomitant to Ca 2+ oscillation after exposure to 7-ketocholesterol. However in a previous study, we showed that ionomycin (calcium ionophore) had no effect on the Nox4 activity in chondrocytes C-20/A4 cells over-expressing Nox4A [18].…”

“…Primary discovered in kidney tissue [13], Nox4 appears to be ubiquitously distributed. Its dysfunction has been linked to several pathologies including hypertension [14], diabetes [15], atherosclerosis [16], cancer [17], osteoarthritis [18] and inflammation [19], and Nox4 represent a potential therapeutic target [20]. Despite its wide distribution, its activation mechanisms at the molecular level are unclear.…”

“…Nox4 is unique among other Nox isoenzymes in that its activity is constitutive and may depend on a specific conformation of the dehydrogenase DH domain that should allow a spontaneous transfer of electrons from NADPH to FAD and to the hemes [24,25]. Data from various studies indicated clearly that Nox4 activity is regulated at the mRNA level, implying that an increase or decrease of ROS production by Nox4 is correlated to an up regulation [9,16,[26][27][28][29][30][31][32][33] or to a decline [34] of Nox4 transcripts. Although it has not been reported yet, post-translational regulation of Nox4 oxidase activity cannot be excluded.…”

Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

“…Oxidative stress is increasingly implicated in the development of atherosclerosis (5) and increased oxidative damage, and elevated levels of DNA strand breaks occur in human atherosclerotic plaques (6). Oxidative stress reduces IGF1R expression and induces VSMC apoptosis in culture (7)(8)(9)(10). Reduced IGF1R expression is also seen within plaques, suggesting that IGF1R-dependent survival regulates apoptosis in vivo (11,12).…”