Calpain is required for macroautophagy in mammalian cells

Abstract

Ubiquitously expressed micro- and millicalpain, which both require the calpain small 1 (CAPNS1) regulatory subunit for function, play important roles in numerous biological and pathological phenomena. We have previously shown that the product of GAS2, a gene specifically induced at growth arrest, is an inhibitor of millicalpain and that its overexpression sensitizes cells to apoptosis in a p53-dependent manner (Benetti, R., G. Del Sal, M. Monte, G. Paroni, C. Brancolini, and C. Schneider. 2001. EMBO J. 20:2702-2714). More recently, we have shown that calpain is also involved in nuclear factor kappaB activation and its relative prosurvival function in response to ceramide, in which calpain deficiency strengthens the proapoptotic effect of ceramide (Demarchi, F., C. Bertoli, P.A. Greer, and C. Schneider. 2005. Cell Death Differ. 12:512-522). Here, we further explore the involvement of calpain in the apoptotic switch and find that in calpain-deficient cells, autophagy is impaired with a resulting dramatic increase in apoptotic cell death. Immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments demonstrate that autophagy is impaired in CAPNS1-deficient cells. Accordingly, the enhancement of lysosomal activity and long-lived protein degradation, which normally occur upon starvation, is also reduced. In CAPNS1-depleted cells, ectopic LC3 accumulates in early endosome-like vesicles that may represent a salvage pathway for protein degradation when autophagy is defective.

Figure 1.

CAPNS1^−/− MEFs lacking calpain activity are defective in autophagosome formation. (A and B) Wild-type and CAPNS1^−/− MEFs were stimulated for 3 h with rapamycin or ceramide and analyzed by immunofluorescence using an anti-LC3–specific antibody and PI to stain nuclei (A) or were lysed and used for Western blot analysis with the same antibody to detect both cytoplasmic LC3I and membrane-associated LC3II (B). Actin was used as a loading control. (B) The ratio between the intensities of LC3II and LC3I bands is indicated below each lane. Bar, 20 μm.

Figure 2.

CAPNS1 depletion is coupled to a block in autophagosome formation in human cells. (A and B) U2OS cells were silenced with control siRNA or CAPNS1 siRNA and, 72 h later, were stimulated with ceramide for 3 h to trigger macroautophagy, as indicated in the figure. Afterward, the cells were fixed and decorated with anti-LC3 and PI (A) or were lysed to perform Western analysis against endogenous LC3 (B). Actin was used as a loading control; anti-CAPNS1 antibody was used to check silencing efficiency. (B) The ratio between the intensities of LC3II and LC3I bands is indicated below each lane. Bar, 20 μm.

Figure 3.

Lysosomal induction and long-lived protein degradation are hampered in CAPNS1-deficient MEFs. Wild-type and CAPNS1 MEFs were incubated with the stimuli indicated, namely rapamycin (rapa), etoposide (ET), EBSS (EBS), serum-free medium (sf), and ceramide (cera), in the presence or absence of 3MA and were trypsinized, stained with LysoTracker green, and analyzed by FACS. (A–D) Representative FACS profiles obtained after LysoTracker staining of wild-type and CAPNS1^−/− MEFs before and after rapamycin treatment. The gate set between 10¹ and 10² separates the cells with high fluorescence from the background population. (E–G) The histograms report the percentage of cells with high LysoTracker staining and represent the mean values obtained in four independent experiments. (H and I) Wild-type and CAPNS1^−/− MEFs were grown for 4 h in amino acid–free medium (H) or in serum-free medium (I) in the presence or absence of the autophagy inhibitor 3MA, and, subsequently, long-lived protein degradation was scored as the percentage of TCA-soluble counts on total radioactivity in a standard protein degradation assay. The histograms report the mean values obtained in four independent experiments. Error bars represent SD. SSC, side scatter.

Figure 4.

Autophagosome formation is hampered in CAPNS1^−/− MEFs. (A and B) The electron micrographs show the ultrastructure of early (A) and late autophagosomes (B) in wild-type MEFs. The cell area on the grid squares was estimated by point counting using negatives taken at 600×. (C) Quantification of autophagosomes in wild-type and CAPNS1^−/− MEFs (knockout) grown in control serum-containing medium (FCS) or induced for 3 h with EBSS (EBS) or rapamycin (Rap). Error bars indicate the SEM. The number of AVi and AVd profiles was counted under the microscope at 12,000× from four to five grid squares for each sample.

Figure 5.

Ectopic LC3 constitutively accumulates in vesicles in CAPNS1^−/− MEFs. (A–C) Wild-type (A and C) and CAPNS1^−/− MEFs (B) were transfected with GFP-rLC3, and, 16 h later, the cells were treated for 3 h with the stimuli indicated. Afterward, the cells were fixed and analyzed by fluorescence microscopy. Representative fields are shown. The different patterns of GFP-LC3 distribution were scored in 250 transfected cells for at least three independent experiments. The mean of the percentage of cells with intensely stained bodies and relative SD are indicated beneath each image. Bar, 20 μm.

Figure 6.

Ectopic LC3 forms specific bodies in CAPNS1-depleted human cells. (A and B) U2OS cells were silenced with a control siRNA (A) or with a CAPNS1 siRNA (B) in combination with a control siRNA or ATG5 siRNA as indicated. After 48 h, the cells were transfected with GFP-hLC3 and, 16 h later, were incubated for 3 h with ceramide or solvent alone as a negative control. They were subsequently fixed and analyzed by fluorescence microscopy. Representative fields are shown. (C and D) A U2OS-derivative cell line stably expressing HcRed-hLC3 (U2OS-LC3) was established. U2OS-LC3 cells were transfected with si-control-GFP (C) or siCAPNS1-GFP (D) expression vectors and, 48 h later, were either treated with ceramide or with solvent alone for 3 h as indicated. Finally, the cells were fixed and analyzed by fluorescence microscopy. The different patterns of GFP-LC3 distribution were scored in 250 transfected cells for at least three independent experiments. Bar, 20 μm.

Figure 7.

Ectopic LC3 bodies are enriched with endosome markers in CAPNS1-depleted cells. (A–C) U2OS-LC3 cells were silenced with a control siRNA or a CAPNS1 siRNA as indicated and, 48 h later, were stimulated for 3 h with ceramide to induce macroautophagy. Pepstatin A was added to inhibit lysosomal activity and freeze any colocalization event between LC3 vesicles and lysosomes. Subsequently, the cells were fixed, decorated with anti–LAMP-2 (A), anti-EEA1 (B), and antitransferrin receptor (TR; C), and analyzed by confocal microscopy. Images of representative fields were taken. The Pearson's correlation coefficient, R, is reported for each merge image. Bars, 20 μm.

Figure 8.

GFP-LC3 bodies in CAPNS1-depleted cells are endosome-like structures. Representative immunoelectron microscopy microphotographs showing the ultrastructure of GFP-LC3 bodies in CAPNS1-depleted U2OS cells. Large gold dots mark GFP-positive structures, whereas small gold dots (indicated by arrowheads) are labeled with anti–LAMP-2.

Figure 9.

CAPNS1^−/− MEFs are more sensitive to apoptosis. (A–D) Wild-type and CAPNS1^−/− MEFs were treated with etoposide (ET), ceramide (cera), EBSS, serum-free medium (sf), and vinblastine (vin) or left in control medium (cont) for 20 h and stained with PI and annexin V–FITC. A set of representative experiments with EBSS is shown. The cells in the bottom right quadrant were scored as apoptotic. (E) The mean values obtained in four independent experiments with all of the stimuli. Error bars represent SD.

Figure 10.

Depletion of CAPNS1 results in increased apoptosis in human cells. U2OS cells were transfected with a siRNA specific for CAPNS1, ATG5, or a combination of the two. An ineffective siRNA was used as a negative control. 48 h later, the cells were shifted to amino acid–free medium for an additional 20 h, stained with PI and annexin-FITC, and analyzed by FACS. (A–F) Results of a set of representative experiments. (G) The mean of the values obtained in four independent experiments. Bars indicate the percentage of annexin-positive cells with high (h) PI staining (top right quadrant in A–F) and low (l) PI staining (bottom right quadrant in A–F). Error bars represent SEM.