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• W2112300479 abstract "Ubiquitously expressed μ- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of μ- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation. Ubiquitously expressed μ- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of μ- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation. Calpains are a family of Ca2+-dependent intracellular cysteine proteases. By cleaving their protein substrates, ubiquitously expressed μ-calpain and m-calpain are implicated in a wide variety of biological functions including cell migration, cell cycle regulation, differentiation, and apoptosis (reviewed in Ref. 1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2305) Google Scholar). Both μ- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes capn1 and capn2, respectively, and a common small 28-kDa regulatory subunit encoded by the capn4 gene. The small subunit is essential to calpain activities, as shown by in vitro biochemical studies where a 25-amino acid truncation at the C terminus abolished all detectable calpain activity (2Elce J.S. Davies P.L. Hegadorn C. Maurice D.H. Arthur J.S. Biochem. J. 1997; 326: 31-38Crossref PubMed Scopus (46) Google Scholar). This provided the rationale for the first reported capn4 knock-out mouse model, which interrupted the coding sequences in exon 9 (3Arthur J.S. Greer P.A. Elce J.S. Biochim. Biophys. Acta. 1998; 1388: 247-252Crossref PubMed Scopus (17) Google Scholar, 4Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (292) Google Scholar) and was predicted to truncate 38 C-terminal amino acids from the small subunit. The resulting hypothetical small subunit was not detectable, the steady-state levels of μ-80, m-80 catalytic subunits were reduced, and no calpain activity was observed (4Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (292) Google Scholar). More recently, we have developed a conditionally targeted capn4 locus by inserting loxP sites into intron 8 and the noncoding region of exon 11. 2Y. Tan, N. Dourdin, C. Wu, T. De Veyra, J. S. Elce, and P. A. Greer, submitted publication.2Y. Tan, N. Dourdin, C. Wu, T. De Veyra, J. S. Elce, and P. A. Greer, submitted publication. This allows conditional knock-out of capn4 by Cre-mediated recombination. Although a hypothetical small subunit protein with a 60-amino acid deletion at the C terminus might still be produced, no detectable small subunit was detected, probably because of destabilization of the hypothetical truncated protein. Expression of the large subunits was also greatly diminished, supporting the proposed role for the small subunit in stabilizing large subunits. As expected, mouse embryonic fibroblasts (MEFs) 3The abbreviations used are: MEF, mouse embryonic fibroblast; ER, endoplasmic reticulum; TG, thapsigargin; TN, tunicamycin; MAP kinase, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ASK1, apoptosis signal-regulating kinase 1; E, embryonic day; CMV, cytomegalovirus; Cre, Cre recombinase; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; AO, acridine orange; IRES, internal ribosome entry site; PARP, polyADP-ribose polymerase; PIPES, 1,4-piperazinediethanesulfonic acid; GFP, green fluorescent protein; TNF, tumor necrosis factor.3The abbreviations used are: MEF, mouse embryonic fibroblast; ER, endoplasmic reticulum; TG, thapsigargin; TN, tunicamycin; MAP kinase, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ASK1, apoptosis signal-regulating kinase 1; E, embryonic day; CMV, cytomegalovirus; Cre, Cre recombinase; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; AO, acridine orange; IRES, internal ribosome entry site; PARP, polyADP-ribose polymerase; PIPES, 1,4-piperazinediethanesulfonic acid; GFP, green fluorescent protein; TNF, tumor necrosis factor. from these knockouts also lacked any detectable ubiquitous calpain activity. 2Y. Tan, N. Dourdin, C. Wu, T. De Veyra, J. S. Elce, and P. A. Greer, submitted publication. Many reports on calpain function are based on using small molecule inhibitors, which lack specificity. In contrast, this genetic knock-out model is completely selective, and therefore provides a powerful tool to address the physiological functions of the ubiquitous calpains. Ubiquitous μ- and m-calpains have been suggested to participate in apoptosis by cleaving either pro-apoptotic or anti-apoptotic proteins like p53, Bcl-2, and Bax, depending on the nature of the stimuli and type of cells involved (6Benetti R. Del Sal G. Monte M. Paroni G. Brancolini C. Schneider C. EMBO J. 2001; 20: 2702-2714Crossref PubMed Scopus (100) Google Scholar, 7Chen M. He H. Zhan S. Krajewski S. Reed J.C. Gottlieb R.A. J. Biol. Chem. 2001; 276: 30724-30728Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 8Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (303) Google Scholar). Among these studies, m-calpain was proposed to play a role in a distinct apoptotic pathway initiated by ER stress (9Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar). The ER plays key roles in protein biosynthesis, modification, folding, and trafficking, and it is also the major pool for calcium storage. Perturbation of ER homeostasis abolishes protein folding, and the consequent accumulation of unfolded proteins in the ER imposes so called ER stress on the cell. The ER membrane residing proteins IRE1, Perk, and ATF6 are ER stress sensors, which are maintained in an inactive monomeric state by binding to the chaperone protein Bip. During ER stress, the release of Bip activates these sensors to convey their protective responses, known as the unfolded protein response (UPR). This includes enhancing the transcription of genes encoding ER chaperones and the attenuation of general protein synthesis (10Ma Y. Hendershot L.M. Cell. 2001; 107: 827-830Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Autophosphorylated IRE1 possesses an endoribonuclease activity that alternatively splices the mRNA encoding the transcription factor XBP-1, thus turning on chaperone genes (11Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2067) Google Scholar). The activated Perk kinase phosphorylates eIF-2α and inhibits general protein synthesis (12Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar). Also, the unfolded proteins trigger ATF6 cleavage by the Site-1 and Site-2 proteases to release its cytoplasmic domain, which then enters the nucleus and activates Bip transcription (13Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar). ER stress can be induced by pharmacological agents such as the calcium ionophore A23187, the Ca2+ pump inhibitor thapsigargin, the N-linked glycosylation inhibitor tunicamycin, the ER to Golgi transport inhibitor brefeldin A, and inhibitors of disulfide bond formation such as dithiothreitol (14Breckenridge D.G. Germain M. Mathai J.P. Nguyen M. Shore G.C. Oncogene. 2003; 22: 8608-8618Crossref PubMed Scopus (643) Google Scholar). Excessive ER stress can trigger cellular apoptosis through the activation of caspase-12, which resides on the outside of ER membrane, and is cleaved and activated during ER stress (15Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). As an initiator caspase, caspase-12 triggers the activation of caspases-9, -7, and -3 in a cytochrome c and Apaf-1-independent manner (16Morishima N. Nakanishi K. Takenouchi H. Shibata T. Yasuhiko Y. J. Biol. Chem. 2002; 277: 34287-34294Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 17Rao R.V. Castro-Obregon S. Frankowski H. Schuler M. Stoka V. del Rio G. Bredesen D.E. Ellerby H.M. J. Biol. Chem. 2002; 277: 21836-21842Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Caspase-12 knock-out cells are resistant to ER stress-induced apoptosis (15Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). In vitro biochemical and calpain inhibitor studies in glial cells suggested that m-calpain is responsible for the cleavage of caspase-12 during ER stress (9Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar). Besides its role in ER stress induced apoptosis, calpain might also participate in a mitochondrial-dependent apoptotic pathway by cleaving and thus releasing apoptosis-inducing factor (AIF) from the inner mitochondrial membrane (18Jayanthi S. Deng X. Noailles P.A. Ladenheim B. Cadet J.L. Faseb. J. 2004; 18: 238-251Crossref PubMed Scopus (240) Google Scholar, 19Polster B.M. Basanez G. Etxebarria A. Hardwick J.M. Nicholls D.G. J. Biol. Chem. 2005; 280: 6447-6454Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 20Liou A.K. Zhou Z. Pei W. Lim T.M. Yin X.M. Chen J. Faseb. J. 2005; 19: 1350-1352Crossref PubMed Scopus (67) Google Scholar). Methamphetamine induced apoptosis of striatal glutamic acid decarboxylase containing neurons through the rapid and simultaneous activation of calpain and caspase-12, and release of AIF and cytochrome c from the mitochondria (18Jayanthi S. Deng X. Noailles P.A. Ladenheim B. Cadet J.L. Faseb. J. 2004; 18: 238-251Crossref PubMed Scopus (240) Google Scholar). Calpain activation was persistent under those conditions, so it might have further participated in other downstream apoptotic signaling events. Surprisingly, the activation of caspase-9 in this system was not synergistically accelerated, but delayed (18Jayanthi S. Deng X. Noailles P.A. Ladenheim B. Cadet J.L. Faseb. J. 2004; 18: 238-251Crossref PubMed Scopus (240) Google Scholar). In vitro studies suggested that calpain-mediated cleavage of an N-terminal AIF domain is required to allow its release from Bid-permeabilized mitochondria (19Polster B.M. Basanez G. Etxebarria A. Hardwick J.M. Nicholls D.G. J. Biol. Chem. 2005; 280: 6447-6454Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Calpains might also mediate cross-talk between mitochondrial and ER apoptotic pathways depending on the nature of the apoptotic stimuli and the cell type. In mouse pancreatic β-islet cells, apoptosis induced by type 2 ryanodine receptor-mediated calcium overloading of mitochondria relies on calpain-10 up-regulation and activation (21Johnson J.D. Han Z. Otani K. Ye H. Zhang Y. Wu H. Horikawa Y. Misler S. Bell G.I. Polonsky K.S. J. Biol. Chem. 2004; 279: 24794-24802Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Interestingly, polymorphisms in calpain-10 are linked to human susceptibility to type II diabetes. The c-Jun N-terminal kinase (JNK) pathway is also activated in response to ER stress by IRE1, a serine/threonine kinase, which gets autophosphorylated during UPR. Activated IRE1 recruits TNF receptor-associated factor 2 (TRAF2), which in turn activates the apoptosis signal-regulating kinase ASK1, which in turn activates the MKK4/JNK cascade (22Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2256) Google Scholar, 23Nishitoh H. Matsuzawa A. Tobiume K. Saegusa K. Takeda K. Inoue K. Hori S. Kakizuka A. Ichijo H. Genes Dev. 2002; 16: 1345-1355Crossref PubMed Scopus (1115) Google Scholar, 24Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Although the precise role of JNK in ER stress is not defined, prolonged JNK activation has pro-apoptotic functions downstream of various cell death-inducing stimuli, including TNFα, H2O2, and DNA-damaging agents (25Deng Y. Ren X. Yang L. Lin Y. Wu X. Cell. 2003; 115: 61-70Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 26Kamata H. Honda S. Maeda S. Chang L. Hirata H. Karin M. Cell. 2005; 120: 649-661Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar). Interestingly, protein-tyrosine phosphatase 1B (PTP1B) knock-out cells, which are not capable of activating the IRE1-JNK pathway after ER stress are resistant to apoptosis (27Gu F. Nguyen D.T. Stuible M. Dube N. Tremblay M.L. Chevet E. J. Biol. Chem. 2004; 279: 49689-49693Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In this study, we investigated the role of the ubiquitous calpains in ER stress. MEFs from conditional capn4 knock-out animals were used to establish a role for calpain in two key ER stress apoptosis pathways. We show that calpain-deficient MEFs were resistant to thapsigargin (TG) and tunicamycin (TN)-induced apoptosis. This resistance correlated with a defect in the activation of the caspase-12, -9, -3 cascade and the ASK1/MKK4/JNK/c-Jun cascade. These observations provide compelling genetic and biochemical evidence for a central role for calpain in the conversion of Ca+2 signals from the stressed ER to the caspase-12 apoptotic pathway and JNK activation. Reagents—Tunicamycin, thapsigargin, propidium iodide (PI), trypan blue and acridine orange (AO) were purchased from Sigma. ER-Tracker Red was from Invitrogen Molecular Probes and Annexin-V-PE/7-aminoactinomycin (7-AAD) was from BD Biosciences/Clontech. Anti-μ-calpain, cytochrome-c, Bcl-2, Bax, BclxL and BclsL antibodies were from Santa Cruz Biotechnology. Antibody against both the large (m-80) and small (28-kDa) subunits of m-calpain was previously described (4Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (292) Google Scholar). The pEGFP-m80 plasmid was previously described (28Larsen A.K. De Veyra T. Jia Z. Wells A. Dutt P. Elce J.S. Protein Expr. Purif. 2004; 33: 246-255Crossref PubMed Scopus (6) Google Scholar). Conditional capn4-targeted Allele—The generation of mice harboring a targeted floxed (loxP-flanked) capn4 allele is described elsewhere.2 Briefly, calpain small subunit coding sequences in exons 9, 10, and 11 were flanked by two loxP sites. An internal ribosome entry site (IRES)-lacZ reporter and PGK promoter-Neo selectable gene cassette were inserted after the coding sequences in exon 11 and before the second loxP site to permit X-gal staining and G418 selection. Cre recombinase-mediated excision of sequences between the loxP sites deleted the intervening sequences as well as the lacZ/Neo cassette. We refer to this allele as capn4PZ/PZ before Cre-mediated excision, and capn4P/P after excision. In order to achieve ubiquitous loss of calpain activity, these mice were crossed with a transgenic line which expresses Cre recombinase under the control of the cytomegalovirus (CMV) promoter, referred to as cmv-cre (29Dupe V. Davenne M. Brocard J. Dolle P. Mark M. Dierich A. Chambon P. Rijli F.M. Development. 1997; 124: 399-410Crossref PubMed Google Scholar). The capn4P/P; cmv-cre embryos derived from the capn4+/P; cmv-cre x capn4PZ/PZ breeding pairs died at approximately embryonic day (E) 11.5. Cell Culture—Primary fibroblast cultures were established from E10.5 embryos. The yolk sacs, heads, and internal organs were isolated and used for genotyping by Southern blot hybridization. Carcasses were treated with 0.25% collagenase in 20% fetal bovine serum in phosphate-buffered saline for 30 min at 37 °C. Cells were then washed twice with media and plated in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. Primary MEFs were used in experiments before the third passage. Spontaneously immortalized MEFs were established as previously described (30Todaro G.J. Wolman S.R. Green H. J. Cell Physiol. 1963; 62: 257-265Crossref PubMed Scopus (61) Google Scholar). Briefly, primary MEFs were seeded in 60-mm plates at a density of 3 × 105 cells/plate and maintained at that density by passage every 3 days. Immortalized MEFs were established between the 13th and 16th passages. Lentivirus-mediated Gene Transduction—Lentiviral vectors (pWPXLD, pWPI) were kindly provided by Dr. Trono at University of Geneva. The IRES sequence was excised from pWPI and inserted before the EGFP sequence in pWPXLD using blunt-end ligation. The murine capn4 cDNA was cloned using RT-PCR (forward primer ATCCGACTCAGCTGCGAT; reverse primer TTGAGCTCTGGCTGCTGA) and inserted at a PmeI site upstream of the IRES. Lentivirus was produced by transfecting 293T cells with pWPXLD and the packaging plasmids pMD2G and pCMV-dR8.74 as described (31Wiznerowicz M. Trono D. J. Virol. 2003; 77: 8957-8961Crossref PubMed Scopus (618) Google Scholar). The multiplicity of infection (MOI) was determined by infecting MEFs with different titrations of virus-containing supernatant and measuring the percentage of GFP-positive cells using flow cytometry. To rescue calpain activity, immortalized capn4P/P; cre MEFs were transduced with lentiviruses encoding recombinant mouse capn4. Apoptosis Analysis—MEFs were seeded at 3 × 105 cells per 60 mm plate and allowed to adhere overnight. On the following day, cells were treated with 3 μg/ml tunicamycin (TN) or 5 μm thapsigargin (TG) for the indicated periods. Mock treated cells were exposed to equivalent concentrations of the vehicle, Me2SO. Apoptosis was measured by 0.4% trypan blue exclusion assays and flow cytometry analysis of PE-conjugated Annexin-V and 7-AAD staining, according to the suppliers instructions. Alternatively, apoptotic cells were quantified by measuring the fraction of cells with a sub-G1 content of PI-staining DNA (32Crissman H.A. Steinkamp J.A. Eur. J. Histochem. 1993; 37: 129-138PubMed Google Scholar). Analysis of Lysosome Stability—Cells were exposed to 5 μg/ml acridine orange (AO) for 15 min under standard culture conditions, then released by trypsin treatment and washed twice with medium. Red fluorescence was measured immediately by flow cytometry. Cells with decreased red fluorescence (pale cells) were gated, and their percentages were determined. Western Blotting—Cell monolayers were washed twice with cold phosphate-buffered saline, then incubated for 15 min on ice in lysis buffer (50 mm HEPES, pH 7.6, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 10 mm 2-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml of leupeptin). Cell lysates were scraped from the dishes, transferred to Eppendorf tubes, and centrifuged at 13,000 rpm for 20 min to remove insoluble material. Protein content was determined using the Bradford method. Samples (20 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed using specific primary antibodies, and detected using horseradish peroxidase-coupled secondary antibodies. Release of Cytochrome c—2 × 106 cells were harvested and washed twice in ice-cold phosphate-buffered saline. The cells were then spun at 200 × g for 5 min. The cell pellet was suspended in 600-μl extraction buffer containing 220 mm mannitol, 250 mm sucrose, 50 mm PIPES-KOH (pH 7.4), 50 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, and protease inhibitors. After 30 min of incubation on ice, cells were treated with a Dounce homogenizer for 80 strokes and spun for 60 min at 14,000 × g. Supernatants were analyzed for cytochrome c release by Western blotting (33Hirpara J.L. Seyed M.A. Loh K.W. Dong H. Kini R.M. Pervaiz S. Blood. 2000; 95: 1773-1780Crossref PubMed Google Scholar). In Vitro Analysis of μ- and m-calpain Activities—Calpain activities in cell extracts were detected by casein zymography in a standard Trisglycine system (34Dourdin N. Bhatt A.K. Dutt P. Greer P.A. Arthur J.S. Elce J.S. Huttenlocher A. J. Biol. Chem. 2001; 276: 48382-48388Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). 40 μg of protein was resolved on 8% non-denaturing polyacrylamide gels containing 1.5 mg/ml casein. Calpain was activated by incubating the gels overnight with 5 mm Ca2+, and its activity was assessed by visualization of the casein-cleared regions of the gel after staining with Coomassie Brilliant Blue. Confocal Fluorescence Microscopy Analysis—To examine m-calpain subcellular localization, NIH3T3 cells were transfected with an m-80-EGFP fusion protein-encoding plasmid (pEGFP-m-80) using Lipofectamine 2000 (Invitrogen). After TG or TN treatment, cells were stained with ER-Tracker Red and imaged by confocal fluorescence microscopy using the green and red channels to visualize m80-EGFP and the ER, respectively. Co-localization was quantified using the Pearson's correlation coefficient, which was calculated using the Image-Pro Plus 5.1 software package (Media Cybernetic Inc). Statistical Analysis—Results were expressed as means ± S.D. of at least three independent experiments. Statistical analysis was performed using Student's t test, with level of significance set at p < 0.05. Capn4P/P Cells Lack Calpain Activity and Have Reduced Expression of Calpain Subunit Proteins—Spontaneously immortalized MEFs were established from capn4+/P; cre and capn4P/P; cre embryos, and calpain activity was analyzed by in vitro casein zymograms (Fig. 1A). As expected, μ- and m-calpain activity was detected in capn4+/P; cre MEFs, but not in capn4P/P; cre cells. However, when the capn4P/P; cre MEFs were transduced with a lentivirus encoding the mouse capn4 cDNA, both μ- and m-calpain activity were restored. Western blotting analysis of these same cell lysates revealed the expected elimination of small subunit expression in capn4P/P; cre MEFs, but we also noted that steady-state levels of both μ-80 and m-80 were dramatically reduced; and this defect was also rescued by transduction with the capn4 lentivirus (Fig. 1B). ER Stress Correlated with Calpain ER Translocation and Activation—Calpain inhibitors have been reported to inhibit ER stress-induced apoptosis by inhibiting caspase-12 activation (9Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar). Calpain is widely distributed in the cell cytosol and is associated with membranes; and it might undergo further concentration at membranous structures and in the nucleus in response to specific signals such as calcium (35Gil-Parrado S. Popp O. Knoch T.A. Zahler S. Bestvater F. Felgentrager M. Holloschi A. Fernandez-Montalvan A. Auerswald E.A. Fritz H. Fuentes-Prior P. Machleidt W. Spiess E. J. Biol. Chem. 2003; 278: 16336-16346Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Calpain has been localized at the plasma membrane, with cytosolic membrane surfaces, and within the lumens of the ER and Golgi apparatus (36Hood J.L. Brooks W.H. Roszman T.L. J. Biol. Chem. 2004; 279: 43126-43135Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In primary MEFs, inhibition of N-linked glycosylation in the ER with tunicamycin (TN), or inhibition of the ER-localized Ca2+-ATPase with thapsigargin (TG) caused an elevation in cytoplasmic Ca2+ levels by about 20%; but no difference was observed between capn4+/PZ; cre and capn4P/P; cre MEFs (data not shown). To track calpain localization during TG/TN-triggered ER stress, we performed confocal fluorescence microscopy analysis of NIH3T3 fibroblasts transfected with an m-80-EGFP fusion protein-encoding plasmid (28Larsen A.K. De Veyra T. Jia Z. Wells A. Dutt P. Elce J.S. Protein Expr. Purif. 2004; 33: 246-255Crossref PubMed Scopus (6) Google Scholar). Recombinant m-80-EGFP was widely dispersed in the cells; however, upon TN or TG treatment, we observed a statistically significant increase in calpain ER localization as assessed by increased spectral overlap of m-80-EGFP and ER-Tracker Red (Fig. 2). This indicated that calpain was recruited to the ER in conjunction with calcium release (Fig. 2B). Calpain large subunits have a half-life of 5 days, but they are quickly degraded after activation (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2305) Google Scholar). We therefore assessed the stability of calpain large subunits after TG or TN treatment as an indication of their degree of activation. In primary capn4+/P; cre MEFs, m-80 subunit levels declined during TG or TN treatment (Fig. 3). Although there was substantially less m-80 subunit in primary capn4P/P; cre MEFs, that amount was further diminished after TM or TG treatment (Fig. 3). The μ-80 subunit was not as easily detected in capn4P/P; cre MEFs; however, in capn4+/P; cre MEFs, we determined that the μ-80 subunit also diminished during TN or TG treatment (Fig. 3). This disappearance of calpain catalytic subunits is consistent with the hypothesis that they are activated and subsequently degraded during ER stress. Calpain-deficient MEFs Are Resistant to Thapsigargin-induced Cell Death—To establish whether calpain is involved in the ER stress pathway, we compared TN or TG-induced apoptosis in MEFs with or without calpain. We first used primary MEFs (P-MEFs) from capn4+/P; cre and capn4P/P; cre sibling embryos to perform these apoptosis studies. Similar results were then obtained using spontaneously immortalized capn4P/P; cre MEFs (I-MEFs) and their lentiviral-capn4 rescued counterparts (Fig. 4). After 3 μg/ml TN or 5 μm TG treatment for 24 h, apoptosis was measured by trypan blue staining of total dead cells (Fig. 4A), or annexin-V and 7-AAD staining of apoptotic cells (Fig. 4B). Fewer dead cells were seen in primary capn4P/P; cre MEFs compared with the primary capn4+/P; cre MEFs in response to either TN or TG treatment. Fewer dead cells were also observed in the immortalized capn4P/P; cre MEFs compared with their capn4 rescued counterparts after TG incubation. Apoptosis was also independently assessed by flow cytometry, where cells with a sub-G1 content of PI-staining DNA were considered apoptotic (Fig. 5). This analysis also showed that calpain deficiency in either primary or immortalized MEFs correlated with resistance to ER stress-induced apoptosis. These observations support a permissive role for calpain in ER stress-induced apoptosis.FIGURE 5DNA staining profiles of cells after ER stress. Apoptotic cells were quantified by measuring the fraction of cells with a sub-G1 content of PI-staining DNA by flow cytometry. A, primary P-capn4P/P; cre and P-capn4+/P; cre MEFs were treated for 24 h with TG or TN, as indicated. B, spontaneously immortalized I-capn4P/P; cre MEFs and their lentivirus rescued counterparts (I-rescue) were treated for 30 h with TG.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Calpain-deficient Cells Display Defective ER Stress-induced Activation of the Caspase-12, -9, -3 Cascade—Caspase-12 plays a pivotal role in ER stress-induced apoptosis (15Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). Like other initiator caspases, activation of pro-caspase-12 requires the cleavage of a short inhibitory peptide. Active caspase-12 can then directly activate downstream caspase-9 independently of cytochrome c release from the mitochondria (9Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar, 16Morishima N. Nakanishi K. Takenouchi H. Shibata T. Yasuhiko Y. J. Biol. Chem. 2002; 277: 34287-34294Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). Active caspase-9 in turn activates the executioner caspase-3, leading to apoptosis. Caspase-12-null cells were resistant to ER stress-induced apoptosis, and in vitro experiments showed that m-calpain could mediate conversion of pro-caspase-12 into an active form by cleavage of the N-terminal pro-peptide regulatory sequence (9Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref P" @default.
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• W2112300479 date "2006-06-01" @default.
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• W2112300479 title "Ubiquitous Calpains Promote Caspase-12 and JNK Activation during Endoplasmic Reticulum Stress-induced Apoptosis" @default.
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