Endoplasmic reticulum stress signaling transmitted by ATF6 mediates apoptosis during muscle development

Abstract

Although apoptosis occurs during myogenesis, its mechanism of initiation remains unknown. In a culture model, we demonstrate activation of caspase-12, the initiator of the endoplasmic reticulum (ER) stress-specific caspase cascade, during apoptosis associated with myoblast differentiation. Induction of ER stress-responsive proteins (BiP and CHOP) was also observed in both apoptotic and differentiating cells. ATF6, but not other ER stress sensors, was specifically activated during apoptosis in myoblasts, suggesting that partial but selective activation of ER stress signaling was sufficient for induction of apoptosis. Activation of caspase-12 was also detected in developing muscle of mouse embryos and gradually disappeared later. CHOP was also transiently induced. These results suggest that specific ER stress signaling transmitted by ATF6 leads to naturally occurring apoptosis during muscle development.

Figure 1.

The ER stress-specific caspase cascade is activated during myoblast differentiation. (A) Induction of either apoptosis or myotube formation. Phase-contrast images of cells are shown. Note that the small round cells in growing culture (GM) are mitotic cells. Bars, 200 μm. (B) Expression of myosin in multinucleated myotubes detected by immunocytochemistry with antimyosin antibody. Bars, 100 μm. (C) Activation of caspase-12 in apoptotic cells examined by Western blot analysis. Arrowheads depict active forms of caspase-12 detected with anti–caspase-12 mAb. D, dead cells; L, live cells. As controls, cells treated with 1 μM thapsigargin (24 h) were also examined. (D) Western blot analysis of caspase-9 and -3 (left). Arrowheads depict active forms. Vimentin cleavage was detected by anti-V1 antibody (Nakanishi et al., 2001). Integrity of mitochondrial transmembrane potential was examined using the JC-1 reagent, which stains intact mitochondria orange (right). Apoptotic cells are indicated by arrowheads. Bars, 20 μm. (E) Suppression of apoptosis in MAGE-3-transfected cells cultured in DM at day 1. Left, phase-contrast; right, antimyosin immunostaining. Bars, 200 μm.

Figure 2.

ER stress signals are generated in C2C12 cells in DM. (A) Induction of BiP in dying (D) and living (L) cells determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acted as loading control. Cells treated with 1 μM thapsigargin (24 h) were also examined as positive controls. (B) Myosin was expressed in both apoptotic cells and differentiating cells. (C) Detection of induction of CHOP by immunocytochemical analysis of differentiating C2C12 cells. Bars, 100 μm. (D) CHOP induction in MAGE-3 transfectants. Bars, 100 μm.

Figure 3.

ATF6 is important in caspase-12 activation during myotube formation. (A) Activation states of ER stress sensors. Western blot analysis of myoblast cells cultured in DM for 24 h. L, live cells; D, dying cells. As positive controls, cells treated with either 2 μg/ml tunicamycin or 1 μM thapsigargin for 24 h were included in the analysis. (B) AEBSF inhibits apoptosis in C2C12 cells in DM. Proliferating cells were pretreated with 400 μM AEBSF or 100 μM p-APMSF for 1 h, and then transferred to DM in the presence of inhibitor for 24 or 48 h. Bars, 200 μm. (C) AEBSF treatment prevents induction of CHOP and myosin. Cells were immunostained by anti-CHOP antibody and antimyosin antibody. Bars: (CHOP) 50 μm; (Myosin) 100 μm.

Figure 4.

Activation of caspase-12 and induction of CHOP in developing muscle tissue. (A) Caspase-12 was induced at the onset of muscle formation at E13.5 in embryo back muscle tissue. (B) Back muscle at E13.5 was specifically stained with antibody to active caspase-12. (C) Higher magnification of images in A and B. The muscle tissue at E13.5 was stained with antibody to caspase-12. Also shown is double-labeling of cells with anti-active caspase-12 antibody (brown) and antimyosin antibody (purple). (D and E) CHOP induction in back muscle tissues at E13.5. Arrows indicate nuclear accumulation of CHOP, whereas arrowheads point to cells with cytoplasmic CHOP. (F) Activation of caspase-12 in the presence of CHOP induction. Anti-p10N antibody, purple; CHOP, brown. Bars: (A, B, and D) 100 μm; (C, E, and F) 10 μm.

Figure 5.

Activation of the ER stress-specific caspase cascade in apoptotic cells in developing muscle tissue at E13.5. (A) Small round cells stained by anti-active caspase-12 antibodies contained condensed nuclei (arrowheads). Nuclei were stained by Gill's modified hematoxylin solution (Merck). (B) Small round cells stained with anti-V1 and anti-active caspase-3 antibodies. Bars, 10 μm.