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. 2014 Jul 11;289(28):19317-30.
doi: 10.1074/jbc.M114.550111. Epub 2014 May 27.

The Mitochondrial Protein NLRX1 Controls the Balance Between Extrinsic and Intrinsic Apoptosis

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Free PMC article

The Mitochondrial Protein NLRX1 Controls the Balance Between Extrinsic and Intrinsic Apoptosis

Fraser Soares et al. J Biol Chem. .
Free PMC article

Abstract

NLRX1 is a mitochondrial Nod-like receptor (NLR) protein whose function remains enigmatic. Here, we observed that NLRX1 expression was glucose-regulated and blunted by SV40 transformation. In transformed but not primary murine embryonic fibroblasts, NLRX1 expression mediated resistance to an extrinsic apoptotic signal, whereas conferring susceptibility to intrinsic apoptotic signals, such as glycolysis inhibition, increased cytosolic calcium and endoplasmic reticulum stress. In a murine model of colorectal cancer induced by azoxymethane, NLRX1-/- mice developed fewer tumors than wild type mice. In contrast, in a colitis-associated cancer model combining azoxymethane and dextran sulfate sodium, NLRX1-/- mice developed a more severe pathology likely due to the increased sensitivity to dextran sulfate sodium colitis. Together, these results identify NLRX1 as a critical mitochondrial protein implicated in the regulation of apoptosis in cancer cells. The unique capacity of NLRX1 to regulate the cellular sensitivity toward intrinsic versus extrinsic apoptotic signals suggests a critical role for this protein in numerous physiological processes and pathological conditions.

Keywords: Apoptosis; Carcinogenesis; Cell Death; Colon Cancer; Innate Immunity; Mitochondria; NLRs; Nod-like Receptor (NLR); Transformation.

Figures

FIGURE 1.
FIGURE 1.
NLRX1 is a glucose-regulated gene that modulates mitochondrial homeostasis. A and B, NLRX1 expression in MEFs treated for 24 h with LPS, hypoxia (1% oxygen), or vesicular stomatitis virus (VSV) (A) or decreased extracellular glucose alone or in combination with 10 mm 2-deoxyglucose (2-DG) (B). C, NLRX1 promoter-driven luciferase activity in HEK293T cells stimulated as in B. D, NLRX1 and SV40 expression by qPCR and immunoblot in primary (P) or SV40-transformed WT and NLRX1-KO MEFs at basal conditions. E, ATP levels in SV40-transformed MEFs treated with 10 mm 2-DG alone or in combination with 10 mm sodium azide. F, qPCR expression of Ki-67 and Cyclin D1 in resting conditions or following 24-h treatment with 10 mm 2-DG or 1 mm glucose. G, measurement of ROS levels in primary or SV40 MEFs at basal conditions or in the presence of rotenone. Data are presented as the mean ± S.D. and are representative of two to four independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.0001; N.D., not detectable). In A and B, error bars represent S.E., and in C–G, error bars represent S.D. CTR, control.
FIGURE 2.
FIGURE 2.
NLRX1 protects transformed MEFs from extrinsic apoptosis. A, cytotoxicity assays measuring the reduction of the tetrazolium dye MTT in primary or SV40-transformed MEFs treated for 24 h with CHX (10 μg/ml) plus TNF (10 ng/ml). B, representative experiments of Annexin V/PI staining of primary and SV40-transformed MEFs treated with CHX (10 μg/ml) plus TNF (10 ng/ml) alone or in combination with Z-VAD-fmk (20 μm). C, caspase-3/7 and caspase-8 activation measured in SV40-transformed MEFs treated for 6 h with CHX (10 μg/ml) plus TNF (10 ng/ml) or staurosporine (ST; 1 μm). D, immunoblot of cleaved caspase-3, cleaved PARP-1, tubulin, and NLRX1 in primary or SV40-transformed MEFs treated for 6 h with CHX ± TNF. E, mitochondria isolated from WT or NLRX1-KO MEFs were incubated for 15 min at 30 °C with increasing concentrations (in nm) of recombinant tBid. Mitochondrial and supernatant fractions were then resolved by SDS-PAGE and immunoblotted with an antibody against cytochrome c. Equal protein loading of the mitochondrial pellet lanes was monitored using an anti-Hsp60 antibody. Data are presented as the mean ± S.D. and are representative of at least three independent experiments (**, p < 0.01; ***, p < 0.0001). Error bars represent S.D. CTRL, control.
FIGURE 3.
FIGURE 3.
NLRX1 promotes intrinsic apoptosis. A and B, immunoblot of cleaved caspase-3, cleaved PARP-1, and tubulin in primary or SV40-transformed MEFs treated with 2-DG (20 mm) (A) or with thapsigargin (3 μm) or the calcium ionophore A23187 (2.5 μm) (B) and CHX (10 μg/ml) ± TNF (10 ng/ml). C and D, Annexin V/PI staining of primary (C) and SV40-transformed (D) MEFs treated with A23187 (2 μm; 18 h) or 2-DG (20 mm; 18 h).
FIGURE 4.
FIGURE 4.
NLRX1-KO cells exhibit normal ER stress but decreased ER stress-induced cell death. A, immunoblot of cleaved caspase-3, cleaved PARP-1, and tubulin in primary or SV40-transformed MEFs treated for 6 or 18 h with increasing dosages of thapsigargin (1, 5, or 10 μm). B, SV40 MEFs were stimulated for 6 h with thapsigargin (1 μm), tunicamycin (1 μg/ml), or A23187 (1 μm), and RT-PCR or qPCR was performed to evaluate the levels of Xbp1 splicing. s, spliced; us, unspliced. C, mRNA expression of Atf3, Chop, and Bip in MEFs stimulated as described in B. Error bars represent S.D.
FIGURE 5.
FIGURE 5.
NLRX1 increases the susceptibility to AOM-induced tumorigenesis. WT and NLRX1−/− mice were sacrificed 6 months after the last AOM administration. A–C, the number of tumors per colon (A), cumulative tumor size per mouse (B), and percentages of tumor size groups (C) were analyzed. D, macroscopic observation of tumors (arrow) in large bowel from AOM-treated WT (top) and NLRX1-KO (bottom) mice. E, representative H&E micrographs of polyps showing adenoma and aberrant crypt foci (ACF) from AOM-treated WT and NLRX1-KO mice, respectively. F, histopathological evaluation of aberrant crypt foci (ACF), adenomas, high grade dysplasia (HGD), and invasive colorectal carcinoma (Inv. Car.) that developed in AOM-treated mice. Error bars represent S.E.
FIGURE 6.
FIGURE 6.
NLRX1 protects mice against DSS colitis and tumorigenesis induced by AOM and DSS. A–E, WT and NLRX1−/− mice received DSS (3%) in drinking water and were examined at day 7 post-treatment. A, histopathological scores. B, representative micrographs of H&E-stained distal colonic sections showing increased crypt damage and ulcerations in DSS-treated NLRX1-KO mice (lower right) compared with DSS-treated WT mice (lower left). C, ELISA measurement of lipocalin-2 levels in feces. D, quantification of apoptotic bodies observed per 10 high power fields (hpf). E, representative H&E-stained distal colonic sections from WT and NLRX1-KO mice showing increased epithelial apoptosis in NLRX1-KO mice compared with WT mice. Apoptotic cells display dark brown-stained nuclei (black arrows). F–N, analysis of WT and NLRX1−/− mice in the AOM-DSS model. Total number of tumors (F) and tumor sizes (G) were analyzed. H, representative micrographs showing polyps (arrow) in large bowels from AOM-DSS-treated mice. I–L, histopathological evaluation displaying scores of inflammation (I); percentages of various tumor grades (aberrant crypt foci (ACF), adenomas, high grade dysplasia (HGD), and invasive colorectal carcinoma (Inv. Car.)) (J); hyperplasia scores (K); and breakdown of the tumors per grade (L). M, representative micrographs of dysplastic colon sections. N, quantitative scoring of neutrophil recruitment. Data are expressed as means ± S.E. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars represent S.E. PMN, polymorphonuclear neutrophil; HPF, high power field.
FIGURE 7.
FIGURE 7.
NLRX1-KO mice exhibit advanced apoptosis and increased proliferation in a short AOM/DSS model. A, schematic illustrating the acute AOM/DSS model. B, quantitative analysis of cleaved caspase-3-positive cells by immunohistochemistry (IHC). C, representative micrographs of colon sections stained for cleaved caspase-3 in WT or NLRX1-KO mice ±AOM/DSS treatment. The left and right panels are enlargements of caspase-3 (Casp-3)-positive cells in WT and NLRX1-KO AOM/DSS-treated mice. D, quantitative analysis of apoptotic bodies by capase-3 immunostaining. E, quantitative analysis of Ki-67 expression measured with immunohistochemistry. F, representative micrographs of colon sections stained for Ki-67 in WT or NLRX1-KO mice ±AOM/DSS treatment. Each data point represents one mouse. Data are expressed as means ± S.E. (**, p < 0.01; ***, p < 0.001). Error bars represent S.E.
FIGURE 8.
FIGURE 8.
Model for NLRX1 modulation of apoptosis. Schematic illustrating NLRX1 regulates the cellular sensitivity toward intrinsic versus extrinsic apoptotic signals. FASL, FAS ligand.

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