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First published online 5 September 2006
doi: 10.1242/jcs.03160

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ER stress-induced apoptosis and caspase-12 activation occurs downstream of mitochondrial apoptosis involving Apaf-1

¹ Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Japan
² Division of Molecular and Cellular Immunoscience, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga, 849-8501, Japan

View larger version (45K): [in a new window]   Fig. 1. Apaf-1-/- MEFs show resistance to ER stress-induced cell death. (A) Cell survival of Apaf-1+/- (WT) and Apaf-1-/- (KO) MEFs in response to ER stress. MEFs were treated with the indicated doses of tunicamycin or thapsigargin for 24 hours and cell survival was determined by Annexin V and propidium iodide (PI) staining with flow cytometry: Annexin V and PI negative population was regarded as viable. Shown are mean + s.d. (B) caspase-3 activation during ER stress. Apaf-1+/- and Apaf-1-/- MEFs treated for indicated hours with 1 µg/ml tunicamycin or 0.1 µM thapsigargin were lysed and analyzed for caspase-3 cleavage. (C) Time course of ER stress-induced cell death of MEFs. MEFs were treated with either 1 µg/ml tunicamycin or 0.1 µM thapsigargin for indicated hours, collected, and analyzed for cell viability as described in A. Shown are mean ± s.d. (D) Transmission electron microscopic analysis of Apaf-1+/- and Apaf-1-/- MEFs. MEFs were treated with 1 µg/ml of tunicamycin for 0, 48 or 72 hours. Experiments were repeated three times with similar results.

View larger version (27K): [in a new window]   Fig. 2. Bcl-XL protects MEFs from ER stress-induced cell death. (A) Cell survival of MEFs overexpressing Bcl-XL. bcl-xl was introduced into MEFs with retrovirus-mediated transduction system. Apaf-1+/- (WT) or Apaf-1-/- (KO) MEFs with or without bcl-xl transduction were treated with tunicamycin or thapsigargin for 24 hours. Cell survival was analyzed as described in Fig. 1A. (B) Caspase-3 activation in MEFs overexpressing Bcl-XL. Cells as in A were analyzed for expression of GRP78 and for proteolytic activation of caspase-3. (C) Time course of ER stress-induced cell death of in Bcl-XL-overexpressing MEFs. Cells as in A were analyzed for cell viability after treatment with either 1 µg/ml tunicamycin or 1 µM thapsigargin for indicated hours. Shown are mean ± s.d. Experiments were repeated three times with similar results. Note that the difference of kinetics of cell death in Apaf-1-/- MEFs from Fig. 1C may be due to the infection of retrovirus.

View larger version (133K): [in a new window]   Fig. 3. Cytochrome c is released from mitochondria in MEFs in response to ER stress. Apaf-1+/- MEFs were treated with either thapsigargin or tunicamycin for 24 hours. Cells were fixed and analyzed for intracellular localization of cytochrome c and BAX by immunofluorescence. Arrowheads show cells in which cytochrome c was released from mitochondria and BAX was translocated into mitochondria.

View larger version (32K): [in a new window]   Fig. 4. Caspase-12 is processed downstream of the apoptosome in MEFs in response to ER stress. (A) Caspase-12 processing during ER stress-induced cell death was analyzed by western blotting. MEFs (Apaf-1+/-; WT or Apaf-1-/-; KO) were treated with tunicamycin (1 µg/ml) with or without Caspase-3 specific inhibitor Ac-DNLD-CHO (100 mM) for the indicated time. Caspase-12 (C12) activation was detected by western blotting. C3, caspase-3. (B) MEFs were introduced with wild or mutated caspase-12 and analyzed for caspase-12 processing by western blotting. (C) Effect of caspase-12 overexpression to ER stress-induced cell death was analyzed by flow cytometry (Annexin V and PI staining). (D) Effect of caspase-12 overexpression on caspase-3 activation was determined by western blotting. (E) Reduced expression of caspase-12 by siRNA was confirmed by western blotting. (F) Apaf-1+/- (WT) or Apaf-1-/- (KO) MEFs with reduced caspase-12 expression (by C12 si-3 in E) were analyzed for their cell survival by flow cytometry. Shown are means ± s.d. Experiments were repeated twice with similar results.

View larger version (77K): [in a new window]   Fig. 5. Loss of Apaf-1 protects the renal epithelium from tunicamycin. (A) Apaf-1+/- (WT) and Apaf-1-/- (KO) adult mice were injected intraperitoneally with 3 µg/gram tunicamycin. After 3 days, mice were sacrificed and kidneys were lysed or fixed for GRP78 expression or histological analyses, respectively. The induction of GRP78 in the kidneys was analyzed by western blotting. (B) Photomicrographs (original magnification, x50; upper panels and x200; lower panels) of kidney sections from Apaf-1+/- or Apaf-1-/- mice after tunicamycin injection. Thin sections were stained with hematoxylin and eosin. (C) TUNEL assay in kidneys from mice 48 hours after injection. Thin sections were stained for TUNEL assay or with DAPI. Experiments were repeated twice with similar results.