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. 2008 Feb 22;283(8):4766-77.
doi: 10.1074/jbc.M706666200. Epub 2007 Dec 11.

Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus

Yongjun Wang  1 , Rajat Singh, Ashish C Massey, Saul S Kane, Susmita Kaushik, Taneisha Grant, Youqing Xiang, Ana Maria Cuervo, Mark J Czaja
Affiliations

Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus

Yongjun Wang et al. J Biol Chem. .

Abstract

Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as macroautophagy has been shown recently to protect against apoptosis. To better define the interplay between these two critical cellular processes, we determined whether inhibition of macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-alpha (TNF-alpha). Atg5-/- mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-alpha in concert with decreased ATP levels. Fas/TNF-alpha treatment failed to up-regulate macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-alpha, Atg5-/- cells were resistant to death from menadione and UV light. In the absence of macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.

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Figures

FIGURE 1
FIGURE 1. Loss of Atg5 sensitizes MEFs to death from Jo2 antibody and TNF-α
Wild-type (WT) and Atg5−/− MEFs were treated with Jo2 antibody (Fas) or TNF-α for various times. A, the percentage cell death was determined by MTT assay at 24 h. Data are from 6 independent experiments (*, p < 0.0001 as compared with wild-type cells). B, cells that were untreated (Con) or treated with Jo2 or TNF-α for 12 h were costained with acridine orange and ethidium bromide, and the percentage of necrotic (nec) and apoptotic (apop) cells was determined by fluorescence microscopy as described under “Experimental Procedures.” Results are from 3 independent experiments performed in duplicate (*, p<0.0001 as compared with the same type of cell death in wild-type cells). C, the percentage cell death was determined by MTT assay after 48 h of treatment. Data are from 4 experiments performed in duplicate (*, p < 0.0001 as compared with wild-type cells).
FIGURE 2
FIGURE 2. Death receptor-induced Atg5−/− cell death results from caspase-dependent apoptosis
A, caspase 8 activity was assayed in wild-type (WT) and Atg5−/− MEFs treated with Jo2 antibody (Fas) for 8 or 12 h. Results are from 4 experiments (*, p < 0.01 as compared with wild-type cells). Con, untreated. B and C, protein was isolated from untreated wild-type and Atg5−/−cells and cells treated with Jo2 (Fas) (B) or TNF-α (C) for the indicated hours. Aliquots of protein were immunoblotted with antibodies for caspase 3 (casp 3), PARP, cIAP2, TRAF2, Atg5, Atg7, Beclin 1, phospho-mTOR (P-mTOR), and mTOR. For Atg5, the band corresponding to the Atg5/12 conjugate protein is shown. Stripped membranes were reprobed with β-actin as a measure of equivalent protein loading among samples. The caspase 3 and PARP cleavage products are indicated by arrows. Immunoblots are representative of 3 independent experiments. D, Atg5−/− cells were pretreated with Me2SO (DMSO) vehicle, 5 (QVD 5) or 10 (QVD 10) µm Q-VD-OPh, or 25 µm IDN-1529 (1529). Cells were then treated with Jo2 antibody (Fas) or TNF-α, and the percentage of cell death was determined by MTT assay at 24 h. Results are from 3 independent experiments (*, p < 0.00001 as compared with Me2SO-reated cells).
FIGURE 3
FIGURE 3. Mitochondrial death pathway activation is increased in Atg5−/− cells after death receptor stimulation
Cytosolic and mitochondrial proteins were isolated from wild-type (Atg5+) and Atg5−/− (Atg5−) cells that were untreated or treated with Jo2 (Fas) (A) or TNF-α (B) and immunoblotted with antibodies for cytochrome c (cyt c), Bid, Bad, Bax, or Bcl-XL. Stripped membranes were reprobed for cytochrome oxidase. Bid cleavage product is indicated by an arrow. C, Atg5−/− cells were infected with adenoviruses that express β-galactosidase (LacZ), Bcl-2, or Bcl-XL. Cells were then treated with Jo2 (Fas) or TNF-α, and the percentage of cell death was determined at 24 h by MTT assay. Data are from 4 independent experiments (*, p < 0.0002). Untreated, wild-type (WT) and knock-out MEFs were stained with Mito Tracker to highlight mitochondria. Top, representative field (bar: 10µm). Bottom, number of mitochondria per cell, average mitochondrial size, and the total cellular area occupied by mitochondria were quantitated. Values are the means of 6–8 fields such as the ones shown (*, p < 0.01 as compared with wild-type cells). E, ATP levels in wild-type and knock-out MEFs untreated (Con) and treated with Jo2 (Fas) for 24 h. Results are from 4 independent experiments (*, p < 0.001 as compared with control cells).
FIGURE 4
FIGURE 4. Jo2 and TNF-α induce changes in the macroautophagic compartment
A and B, MEFs treated with Jo2 (Fas), TNF-α, or menadione (Men) for 4 (A) or 24 h (B) were subjected to immunoblotting with an antibody for LC3. Where indicated, 20mm ammonium chloride and 100µm leupeptin (NH4Cl/Leup) were added to the incubation medium. Stripped membranes were reprobed for β-actin. The unconjugated (I) and conjugated (II) LC3 forms are indicated. The LC3-II to LC3-I ratio (II:I) and the -fold increase in LC3-II in the presence of the protease inhibitors (+PI:None) are shown. Values are the means from densitometric scans of immunoblots from 3 independent experiments. Con, untreated. C and D, immunofluorescence staining for LC3 in the same cells. C, representative fields are shown (bar: 5µm). D, the numbers of fluorescent punctate structures per cell (left) and the average size of the LC3-positive puncta (right) were determined in 20 cells/experiment. Data are from 3 independent experiments (*, p < 0.05 between control and Fas or TNF-α treatment).
FIGURE 5
FIGURE 5. Loss of Atg5 protects against toxicity from menadione and UV light but not from staurosporine- and endoplasmic reticulum stress-induced death
Wild-type (WT) and Atg5−/− MEFs were treated with the indicated µm concentrations of menadione (A), energy levels of UV light (B), and µm concentrations of staurosporine (C) or tunicamycin (D). The percentage cell death was determined by MTT assay at 6 h for staurosporine and at 24 h for the other treatments. The data are from 3–4 independent experiments (*, p < 0.001 as compared with wild-type cells).
FIGURE 6
FIGURE 6. CMA up-regulation protects against menadione toxicity
A, total rates of protein degradation in wild-type MEFs untreated (Con) or treated with menadione (Men) were calculated as described under “Experimental Procedures.” Data are from 3 independent experiments with triplicate wells for each experiment (*, p< 0.05 as compared with control). B, macroautophagic activity in wild-type MEFs was calculated as the percentage of lysosomal degradation sensitive to inhibition by 3-MA in the same experiments (*, p<0.01 as compared with control). C, intracellular distribution of LAMP-2A/hsc70 enriched lysosomes in wild-type (WT) and Atg5−/− MEFs untreated or treated with menadione. Top, representative image of double labeled cells. Bottom, representative image of the distribution of hsc70 puncta (bar: 5 µm). Right, the percentage of LAMP-2A and hsc70 colocalization and the distance of hsc70-containing lysosomes from the nucleus. Data are from the quantification of six different fields (average 20 cells total) in 2 independent experiments (*, p<0.01 as compared with untreated wild-type cells). D, LAMP-2A RNA interference NIH3T3 cells were treated with menadione, and the percentage of cell death was determined by MTT assay at 24 h. Results are from 4 independent experiments (*, p < 0.001 as compared with wild-type cells).
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