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. 2010 Dec 13;191(6):1113-25.
doi: 10.1083/jcb.201006121. Epub 2010 Dec 6.

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

Gang Li  1 , Christopher Scull, Lale Ozcan, Ira Tabas
Affiliations

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

Gang Li et al. J Cell Biol. .

Abstract

Endoplasmic reticulum (ER)-induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress-apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA-dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.

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Figures

Figure 1.
Figure 1.
ER-stressed macrophages undergo oxidative stress, which is dependent on CHOP, calcium, and CaMKII. (A) Peritoneal macrophages from Chop+/+ (WT) and Chop−/− mice were incubated for 15 h without sterol (Con), under cholesterol-loading conditions (CHOL), or with 7-ketocholesterol (7KC). Intracellular peroxide accumulation was then assayed by DCF fluorescence. Three fields for each sample were quantified and expressed as a percentage of DCF-positive cells. (B) Macrophages were pretreated for 1 h with 5 µM BAPTA-AM or with equivalent volumes of vehicle (Veh). The cells were then incubated for 15 h under control or cholesterol-loading conditions and also with BAPTA-AM or vehicle control as indicated. Intracellular peroxide accumulation and apoptosis were assayed by DCF fluorescence (green) and Alexa Fluor 594–conjugated annexin V (red), respectively. Bar, 20 µm. (C) Macrophages were pretreated for 1 h in the absence or presence of 5 µM of the CaMKII inhibitor KN93 or the inactive analogue KN92, followed by incubation for 14 h without sterol under cholesterol-loading conditions or with 7-ketocholesterol. DCF fluorescence and annexin V staining were then assayed. (D) Macrophages were transfected with two different Camk2g siRNA constructs. After 72 h, the cells were incubated for 30 h under control or cholesterol-loading conditions and then assayed for DCF fluorescence and annexin V staining. Bars with the same symbols are not significantly different from each other, whereas bars with different symbols are significantly different from each other. n = 3 for each experimental group. scrRNA, scrambled RNA. Data are presented as means ± SEM.
Figure 2.
Figure 2.
Oxidative stress and apoptosis in ER-stressed macrophages are dependent on NOX2. (A and B) Macrophages were transfected with Nox2 siRNA and, after 72 h, were incubated without sterol (Con), under cholesterol-loading conditions (CHOL), or with 7-ketocholesterol (7KC). The cells were assayed for Nox2 mRNA by RT-QPCR after 8 h (A) and DCF fluorescence and annexin V staining after 30 h (B). (C) Macrophages from WT or Nox2−/− mice were incubated for 14 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol and then assayed for DCF and annexin V staining. (D) Macrophages from WT or Nox2−/− mice were incubated under cholesterol-loading conditions for 24 h or with 7-ketocholesterol for 20 h and then assayed for apoptosis by TUNEL staining. Bars with the same symbols are not significantly different from each other, whereas bars with different symbols are significantly different from each other. scrRNA, scrambled RNA. n = 3 for each experimental group. Data are presented as means ± SEM.
Figure 3.
Figure 3.
Nox2 induction by ER stress is dependent on CaMKII, ERO1α, IP3R1, and JNK. (A, left) Macrophages were pretreated for 1 h in the absence or presence of 5 µM of the CaMKII inhibitor KN93, the inactive analogue KN92, or vehicle control (Veh), followed by incubation for 8 h without sterol (Con) or under cholesterol-loading conditions (CHOL). Nox2 mRNA was then measured by RT-QPCR. (A, right) Peritoneal macrophages from WT or Camk2g−/− mice were incubated for 8 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol (7KC) and then assayed for Nox2 mRNA. (B) Macrophages were transfected with scrambled RNA (scrRNA) or Ero1a siRNA, which, after 72 h, led to an ∼50% decrease in ERO1α expression as assessed by immunoblotting (Li et al., 2009). The cells were then incubated an additional 8 h without sterol or with 7-ketocholesterol and then assayed for Nox2 mRNA. (C) Macrophages were transfected with scrambled RNA or Ip3r1 siRNA. After 72 h, IP3R1 expression was decreased by 60% as assessed by immunoblotting (Li et al., 2009). The cells were then incubated without sterol or under cholesterol-loading conditions for an additional 8 or 30 h and then assayed for Nox2 mRNA or DCF fluorescence, respectively. (D, left) Macrophages were pretreated for 1 h with 10 µM of the JNK inhibitor SP600125 or vehicle control and then incubated for 8 h without sterol or under cholesterol-loading conditions, also in the absence or presence of SP600125. (D, right) WT or Jnk2−/− macrophages were incubated under control conditions or for 4 or 8 h under cholesterol-loading conditions. Nox2 mRNA was then assayed. (E) Macrophages were pretreated for 1 h with the JNK inhibitor SP600125 (SP) or vehicle control and then incubated for 15 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol, also in the absence or presence of SP600125. DCF fluorescence and annexin V staining were then assayed. Bars with the same symbols are not significantly different from each other, whereas bars with different symbols are significantly different from each other. n = 3 for each experimental group. Data are presented as means ± SEM.
Figure 4.
Figure 4.
Evidence for CHOP amplification through NOX/oxidative stress–mediated activation of PKR. (A) Macrophages from WT or Nox2−/− mice were incubated under cholesterol-loading conditions (CHOL) or with 7-ketocholesterol (7KC) for the indicated times. Lysates were then immunoblotted for phospho-CaMKII (p-CaMKII), total CaMKII (T-CaMKII), CHOP, phospho-PERK (p-PERK), phospho-PKR (p-PKR), and GAPDH. (B) Macrophages from WT or Nox2−/− mice were incubated under cholesterol-loading conditions for 8 h (ATF4 experiment) or 9 h (XBP1 experiment). Nuclei were isolated and immunoblotted for ATF4 and nucleophosmin (Np) loading control, or RNA was extracted and assayed for spliced and unspliced Xbp1 and Gapdh loading control. (C) Macrophages were transfected with scrambled RNA (scrRNA) or Pkr siRNA. After 72 h, the cells were incubated for the indicated times under cholesterol-loading conditions. Lysates were then immunoblotted for PKR, phospho-eIF2α (P-eIF2α), CHOP, and β-actin loading control. (D) Macrophages were transfected with scrambled RNA or Pkr siRNA. After 72 h, the cells were incubated for an additional 8 h without sterol (Con) or with 7-ketocholesterol and then assayed for Nox2 mRNA. The data are displayed as Nox2 mRNA levels relative to those for the control scrambled RNA group. (E, left) Macrophages from WT, Chop+/−, or Chop−/− mice were incubated for 14 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol and then assayed for annexin V staining. (E, right) Macrophages were transfected with scrambled RNA or Pkr siRNA. After 72 h, the cells were incubated for an additional 30 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol and then assayed for DCF fluorescence and annexin V staining. In this graph, * indicates P < 0.05 and ** indicates P < 0.01. (F) Peritoneal macrophages were pretreated in the absence or presence of 5 µM of the antioxidant N-acetylcysteine (NAC). The cells were then incubated under cholesterol-loading conditions or with 7-ketocholesterol for the indicated time periods, also in the absence or presence of NAC. Lysates were then immunoblotted for phospho-PKR, CHOP, and β-actin. n = 3 for each experimental group. Data are presented as means ± SEM.
Figure 5.
Figure 5.
NOX is necessary for activation of IP3R and the mitochondrial pathway of apoptosis in ER-stressed macrophages. (A, left) Macrophages from WT or Nox2−/− mice were incubated for 6 h under cholesterol-loading conditions (CHOL) and then assayed for IICR, as reflected by the post-ATP increment in the area under curve for ATP-induced calcium release (n = 30 cells). (A, right) To assess ER luminal calcium stores, the cells were treated with 2 µM thapsigargin. The bar graph shows the mean peak amplitude of the postthapsigargin Fluo-3 response. (B) Macrophages from WT or Nox2−/− mice were incubated for 2 h under control (Con) or cholesterol-loading conditions. 10 µM Rhod-2 was then added to the media, and the cells were incubated on ice for 1 h at 4°C. Next, the cells were rinsed to remove extracellular Rhod-2 and incubated for an additional 5 h, also under control or cholesterol-loading conditions. At the end of the incubation period, the cells were visualized using confocal microscopy and imaged as described in Materials and methods. Fluorescence intensity for ∼100 cells was measured for each treatment group. (C) Macrophages from WT or Nox2−/− mice were incubated for 14 h under control or cholesterol-loading conditions and then assayed for MitoTracker red staining as described in Materials and methods. (D) Macrophages were pretreated for 1 h with 0.5 µM NAC, followed by incubation under control or cholesterol-loading conditions for 14 h, also with or without NAC. The cells were then stained with MitoTracker red CMXRos and imaged and quantified as in C. (E) Macrophages from WT or Nox2−/− mice were incubated for 8 h under control or cholesterol-loading conditions. Cytosolic and mitochondrial fractions were assayed for cytochrome c (Cyto C), GAPDH (cytosolic marker), and prohibitin (mitochondrial marker). For all graphs, bars with the same symbols are not significantly different from each other, whereas bars with different symbols are significantly different from each other. n = 3 for each experimental group. Bars, 10 µm. Data are presented as means ± SEM.
Figure 6.
Figure 6.
NOX2 deficiency protects against ER stress–induced renal cell apoptosis, renal dysfunction, and CHOP induction. (A–C) WT, Camk2g−/−, and Nox2−/− mice were injected i.p. with 1 mg/kg tunicamycin (TUN) or vehicle (Veh) control. The kidneys were harvested 40 h later and stained using anti-NOX2 (A), DHE (superoxide accumulation; B), or TUNEL (apoptosis; C). All sections were also stained with DAPI to visualize nuclei. Bars, 20 µm. The bar graphs show quantification of the mean fluorescent intensities from three mice in each group. (D) Serum creatinine levels and urine albumin levels (normalized to urine creatinine) were determined for all groups of mice. (E) Kidney lysates were assayed for CHOP and β-actin by immunoblotting. For A–D, bars with the same symbols are not significantly different from each other, whereas bars with different symbols are significantly different from each other. n = 3 for each experimental group. Data are presented as means ± SEM.
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