Abstract: Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of multiple diseases. However, the in vivo signaling pathways are still not fully understood. In this report, through the use of genetically deficient mouse embryo fibroblasts (MEFs) and their matched wild-type controls, we have demonstrated that the mitochondrial apoptotic pathway mediated by Apaf-1 is an integral part of ER stress-induced apoptosis and that ER stress activates different caspases through Apaf-1-dependent and -independent mechanisms. In search of the molecular link between ER stress and the mitochondrial apoptotic pathway, we have discovered that in MEFs, ER stress selectively activates BH3-only proteins PUMA and NOXA at the transcript level through the tumor suppressor gene p53. In p53-/- MEFs, ER stress-induced apoptosis is partially suppressed. The p53-independent apoptotic pathway may be mediated by C/EBP homologous protein (CHOP) and caspase-12, as their activation is intact in p53-/- MEFs. In multiple MEF lines, p53 is primarily nuclear and its level is elevated upon ER stress. To establish the role of NOXA and PUMA in ER stress-induced apoptosis, we have shown that, in MEFs deficient in NOXA or PUMA, ER stress-induced apoptosis is reduced. Reversibly, overexpression of NOXA or PUMA induces apoptosis as evidenced by the activation of BAK and caspase-7. Our results provide new evidence that, in MEFs, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic pathway, and both contribute to ER stress-induced apoptosis. Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of multiple diseases. However, the in vivo signaling pathways are still not fully understood. In this report, through the use of genetically deficient mouse embryo fibroblasts (MEFs) and their matched wild-type controls, we have demonstrated that the mitochondrial apoptotic pathway mediated by Apaf-1 is an integral part of ER stress-induced apoptosis and that ER stress activates different caspases through Apaf-1-dependent and -independent mechanisms. In search of the molecular link between ER stress and the mitochondrial apoptotic pathway, we have discovered that in MEFs, ER stress selectively activates BH3-only proteins PUMA and NOXA at the transcript level through the tumor suppressor gene p53. In p53-/- MEFs, ER stress-induced apoptosis is partially suppressed. The p53-independent apoptotic pathway may be mediated by C/EBP homologous protein (CHOP) and caspase-12, as their activation is intact in p53-/- MEFs. In multiple MEF lines, p53 is primarily nuclear and its level is elevated upon ER stress. To establish the role of NOXA and PUMA in ER stress-induced apoptosis, we have shown that, in MEFs deficient in NOXA or PUMA, ER stress-induced apoptosis is reduced. Reversibly, overexpression of NOXA or PUMA induces apoptosis as evidenced by the activation of BAK and caspase-7. Our results provide new evidence that, in MEFs, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic pathway, and both contribute to ER stress-induced apoptosis. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; Etop, etoposide; FITC, fluorescein isothiocyanate; MEFs, mouse embryo fibroblasts; PBS, phosphate-buffered saline; PI, propidium iodide; TG, thapsigargin; Tun, tunicamycin; FACS, fluorescence-activated cell sorter; WT, wild-type; CMV, cytomegalovirus; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PUMA, p53-up-regulated modulator of apoptosis; CHOP, C/EBP homologous protein; BH3, BCL-2 homology domain 3.2The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; Etop, etoposide; FITC, fluorescein isothiocyanate; MEFs, mouse embryo fibroblasts; PBS, phosphate-buffered saline; PI, propidium iodide; TG, thapsigargin; Tun, tunicamycin; FACS, fluorescence-activated cell sorter; WT, wild-type; CMV, cytomegalovirus; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PUMA, p53-up-regulated modulator of apoptosis; CHOP, C/EBP homologous protein; BH3, BCL-2 homology domain 3. is the site for synthesis, folding, modification, and trafficking of secretory and cell-surface proteins. As a major intracellular calcium storage compartment, the ER also plays a critical role toward maintenance of cellular calcium homeostasis. Disruption of these physiological functions by ER stress has been implicated in a wide variety of human diseases, including Alzheimer disease, Parkinson disease, neuronal damage by ischemia, prion disease, cystic fibrosis, and diabetes mellitus (1Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1072) Google Scholar). ER stress could also be elicited in the cell culture system by pharmacological agents including tunicamycin (Tun), a protein N-glycosylation inhibitor; brefeldin A (BFA), which blocks protein transport from ER to Golgi; and thapsigargin (TG), which blocks ER uptake of calcium by inhibiting the sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) (2Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). A variety of ER stresses result in unfolded protein accumulation, which triggers the unfolded protein response (1Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1072) Google Scholar, 3Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1912) Google Scholar). For survival, the cells induce ER chaperone proteins to alleviate protein aggregation, transiently attenuate translation, and activate the proteasome machinery to degrade misfolded proteins. Nonetheless, under severe and prolonged ER stress, unfolded protein response activates unique pathways that lead to cell death through apoptosis (4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1284) Google Scholar). Currently, several pathways have been directly implicated in ER stress-induced apoptosis. For example, the transcription factor CHOP/GADD153 is induced by ER stress at the transcript level, which sensitizes cells to ER stress through down-regulating BCL-2 and activating GADD34 and ERO1α, an ER oxidase (5Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1441) Google Scholar, 6McCullough K.D. Martindale J.L. Klotz L.O. Aw T.Y. Holbrook N.J. Mol. Cell. Biol. 2001; 21: 1249-1259Crossref PubMed Scopus (1547) Google Scholar). ER stress also activates the ER transmembrane protein kinases type I ER membrane protein kinase (IRE1) and PKR-like ER kinase (PERK), which have been implicated in the activation of the pro-apoptotic c-Jun NH2-terminal kinase (JNK) (7Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar, 8Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2256) Google Scholar). Furthermore, ER stress leads to proteolytic cleavage of caspase-12 (C-12) in mouse and caspase-4 (C-4) in human, both of which localize to the cytoplasmic side of the ER membrane (9Hitomi J. Katayama T. Eguchi Y. Kudo T. Taniguchi M. Koyama Y. Manabe T. Yamagishi S. Bando Y. Imaizumi K. Tsujimoto Y. Tohyama M. J. Cell Biol. 2004; 165: 347-356Crossref PubMed Scopus (726) Google Scholar, 10Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). Evidence is also emerging that there is cross-talk between the ER and the mitochondria. The mitochondria-initiated apoptotic pathway is a major one in mammalian cells and is tightly regulated by BCL-2 family proteins. BAX and BAK are pro-apoptotic members activated by a variety of apoptotic stimuli, leading to oligomerization and insertion into the mitochondrial outer membrane to release cytochrome c (4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1284) Google Scholar, 11Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (393) Google Scholar, 12Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (961) Google Scholar). Cytochrome c binds to Apaf-1 and caspase-9 (C-9), resulting in the activation of C-9 and the subsequent activation of caspase-3 (C-3) and caspase-7 (C-7) and ultimately cell death. Recent evidence reveals that BAX/BAK can also localize to the ER and are activated in response to ER stress, leading to calcium depletion and murine caspase 12 (C-12) activation (13Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1213) Google Scholar, 14Zong W.X. Li C. Hatzivassiliou G. Lindsten T. Yu Q.C. Yuan J. Thompson C.B. J. Cell Biol. 2003; 162: 59-69Crossref PubMed Scopus (496) Google Scholar). In MEFs deficient in BAX or BAK, ER stress-mediated C-12 cleavage is abolished but can be restored by the introduction of ER-targeted BAK. However, because BAX/BAK can activate apoptosis from both the mitochondria and the ER, it remains to be determined how dependent the mitochondria-initiated apoptotic pathways are on ER stress-induced apoptosis in vivo. Also, this raises the important issue regarding the molecular links between ER stress and initiation of the mitochondrial apoptotic pathways. In mitochondria-initiated apoptosis, the activation of BAX/BAK involves members of the BH3-only BCL-2 family proteins, which are essential initiators of apoptotic cell death. This large group of proteins share only the BH3 domain, which is used for binding to the anti-apoptotic members of the BCL-2 family and for inducing apoptosis (15Huang D.C. Strasser A. Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Puthalakath H. Strasser A. Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (620) Google Scholar). Among this group of proteins, only a subset is under stringent transcriptional control. Interestingly, one such protein, PUMA, has recently been identified as strongly inducible by ER stress in human neuroblastoma cells and may contribute to ER stress-induced apoptosis in human colon cancer cells (17Reimertz C. Kogel D. Rami A. Chittenden T. Prehn J.H. J. Cell Biol. 2003; 162: 587-597Crossref PubMed Scopus (319) Google Scholar). PUMA is both a target and mediator of p53-mediated apoptosis. In this report, through the use of genetically deficient MEFs and their matched wild-type controls, we investigated the dependence of ER stress-mediated caspase activation and apoptosis on the mitochondria in vivo. In search of regulators of ER stress activation of BAK/BAX in addition to PUMA, we identified NOXA as a novel BH3-only protein that is activated by ER stress at the transcript level. We further showed that the induction of Noxa and Puma by ER stress in MEFs is largely dependent on the tumor suppressor gene p53 and both proteins contribute to ER stress-induced apoptosis. In MEFs, p53 is primarily nuclear, and its level is elevated in MEFs upon ER stress. Further, ER stress-induced apoptosis is partially suppressed in p53-/- MEFs, whereas the activation of C-12 and the induction of CHOP remain intact in p53-/- MEFS, suggesting both p53-dependent and -independent pathways. Our results provide new evidence that, in addition to PUMA, p53 and NOXA are novel components of the ER stress-induced apoptotic pathways. The relationship of these novel findings in MEFs to previous results obtained with other established cell lines is discussed. Cell Culture—Paired wild-type (WT Apaf-1+/+) and Apaf-1-/- MEFs (18Honarpour N. Du C. Richardson J.A. Hammer R.E. Wang X. Herz J. Dev. Biol. 2000; 218: 248-258Crossref PubMed Scopus (179) Google Scholar) were provided by Dr. X. Wang (University of Texas Southwestern Medical Center, Dallas, TX). Paired early passage WT (Puma+/+) and Puma-/- MEFs (19Jeffers J.R. Parganas E. Lee Y. Yang C. Wang J. Brennan J. MacLean K.H. Han J. Chittenden T. Ihle J.N. McKinnon P.J. Cleveland J.L. Zambetti G.P. Cancer Cell. 2003; 4: 321-328Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar) were provided by Dr. G. P. Zambetti (St. Jude Children's Hospital, Memphis, TN). Noxa-/- MEFs (20Villunger A. Michalak E.M. Coultas L. Mullauer F. Bock G. Ausserlechner M.J. Adams J.M. Strasser A. Science. 2003; 302: 1036-1038Crossref PubMed Scopus (1076) Google Scholar) were originally provided to Dr. G. Chinnadurai (St. Louis University, St. Louis, MO) by Dr. A. Strasser (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). Paired WT (p53+/+) and p53-/- MEFs (21Schuler M. Maurer U. Goldstein J.C. Breitenbucher F. Hoffarth S. Waterhouse N.J. Green D.R. Cell Growth & Differ. 2003; 10: 451-460Crossref Scopus (95) Google Scholar) were provided by Dr. D. Green (La Jolla Institute for Allergy and Immunology, San Diego, CA). The fibroblasts and 293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, l-glutamine and antibiotics. All cells were plated 1 day prior to drug treatment. Plasmids—The full-length murine Noxa cDNA was made by reverse-transcribed PCR using total RNA from WT MEFs and the following primers: sense, 5′-TTC TGA GAT GCC CGG GAG AA-3′ and antisense, 5′-GGG AGG TCC CTT CTT GCA AA-3′. The PCR product was directly subcloned into PCR2.1 vector using the TA cloning kit (Invitrogen) and was verified by DNA sequencing. For Northern blots, the cDNA probe was purified after digesting with EcoRI. For expression plasmids, a full-length murine Noxa cDNA flanked with BamHI and EcoRI restriction sites was generated using PCR2.1-Noxa as a template and the following primers: sense, 5′-GCG GGA TCC TGC CCG GGA GAA AGG CGC-3′ and antisense, 5′-GCG GAA TTC TCA GGT TAC TAA ATT GAA GAG-3′. The cDNA was then cloned into pEGFP-C2 (BD Biosciences) to generate pEGFP-Noxa. PUMA expression vector pCMV-Puma (22Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1839) Google Scholar) was provided by Dr. K. H. Vousden (Beatson Institute for Cancer Research). Antibodies and Reagents—Detection of protein expression was performed by standard immunoblotting techniques using primary antibodies against C-3 (rabbit polyclonal antiserum, Cell Signaling), C-7 (mouse IgG1, Pharmingen), C-12 (rabbit polyclonal antiserum, Cell Signaling), CHOP (B3, mouse IgG1, Santa Cruz Biotechnology), GFP (mouse IgG, BD Biosciences), GRP78 (rabbit polyclonal antiserum, Stressgene), NOXA (M16, goat anti-mouse polyclonal antiserum, Santa Cruz Biotechnology.), p53 (Pab241, mouse IgG2a for MEF immunostaining, Calbiochem; Pab240, mouse IgG1, for Western blot, Santa Cruz Biotechnology.), PUMAα (N-terminal, rabbit polyclonal antiserum for mouse cells, Sigma; N-20, goat polyclonal antiserum for human cells, Santa Cruz Biotechnology.), and β-actin (AC15, mouse IgG, Sigma). ER stress inducers TG, Tun, and BFA were purchased from Sigma. Etoposide (Etop) was from Calbiochem. Detection of BAK Activation—Analysis of the conformational changes of BAK was performed as described previously (14Zong W.X. Li C. Hatzivassiliou G. Lindsten T. Yu Q.C. Yuan J. Thompson C.B. J. Cell Biol. 2003; 162: 59-69Crossref PubMed Scopus (496) Google Scholar). 293 cells were transfected with pEGFP or pEGFP-Noxa. Forty-eight hours later, a portion of the cells was used for GFP detection. The rest of the cells were fixed in 0.25% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min. The cells were washed three times with PBS and incubated with 1:50 anti-mouse IgG1 (BD Biosciences) or anti-BAK (AM03; Oncogene Research Products) in 100 μg/ml digitonin (Sigma) in PBS for 30 min. After being washed with PBS three times, the cells were incubated with 1:100 fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ig for 30 min. The cells were subjected to analysis by flow cytometry (FACstar; BD Biosciences). Apoptosis Assays—Cell apoptosis of MEFs was assessed by flow cytometry after staining with annexin-V-FITC (Pharmingen) and propidium iodide (PI, Roche Applied Science) as described previously (23Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). In transient transfection assays, 293 cells were transfected with pEGFP, pEGFP-Noxa, pCMV-Puma alone, or in combination using Polyfect (Qiagen). The final concentration of the plasmid was adjusted to the same amount using pCDNA3 (Invitrogen). Forty-eight hours later, the cells were stained with phycoerythrin-conjugated annexin-V and 7-amino-actinomycin (7-AAD). The apoptosis of GFP-positive cells was analyzed by flow cytometry. For kinetics, cell apoptosis was assessed by flow cytometry and cell cycle analysis following permeabilization and staining with PI. Essentially, ER-stressed cells were trypsinized, washed in ice-cold PBS, and fixed in -20 °C 70% ethanol for 1 h at 4 °C. The cells were stored in -20 °C before preparation for analysis. The fixed cells were washed with cold PBS and treated with DNase-free RNase (200 units/ml, Roche Applied Science) for 30 min at 37 °C. Cellular DNA was stained with 50 μg/ml PI for at least 15 min at room temperature. The cells were analyzed by flow cytometry. Results represent the mean of triplicate determinations in which a minimum of 10,000 cells were assayed for each determination. Any sub-G1 population was counted as apoptotic cells. Northern Blot Analysis—Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's procedure. Ten micrograms of total RNA per sample was subjected to Northern blot analysis, which was carried out as previously described (24Hong M. Luo S. Baumeister P. Huang J.M. Gogia R.K. Li M. Lee A.S. J. Biol. Chem. 2004; 279: 11354-11363Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Probes were made by reverse-transcribed PCR using total RNA from WT MEFs and the following primers: BimL sense, 5′-GCA CCC ATG AGT TGT GAC AA-3′ and antisense, 5′-TCA ATG CCT TCC CAT ACC AG-3′; Bnip3 sense, 5′-GCT CCC AGA CAC CAC AAG AT-3′ and antisense, 5′-CAA GCC AAT GGC CAG CAG AT-3′; Dp-5 sense, 5′-GGA GGA AGC TGG TTC CTG TT-3′ and antisense, 5′-CCC ACC GGT CCA TGT AAG TT-3′; Nix sense, 5′-GAG ATG CAT ACC AGC AGG GA-3′ and antisense, 5′-CAC TTC ACA GGC CAC ACG AA-3′. The PCR products were subcloned into PCR2.1 and were verified by DNA sequencing. The cDNA probes were purified after digesting with EcoRI. Probes for Puma and Spike were prepared from cDNA clones (IMAGE identification numbers 6310857 and 2811290, respectively, Open Biosystems) after digesting with EcoRI/BamHI and EcoRI/HindIII, respectively. The probe for human Bik (25Mathai J.P. Germain M. Marcellus R.C. Shore G.C. Oncogene. 2002; 21: 2534-2544Crossref PubMed Scopus (109) Google Scholar) was a gift from Dr. G. C. Shore (McGill University, Montreal, Quebec, Canada), and the probe for human Chop (26Carlson S.G. Fawcett T.W. Bartlett J.D. Bernier M. Holbrook N.J. Mol. Cell. Biol. 1993; 13: 4736-4744Crossref PubMed Scopus (188) Google Scholar) was a gift from Dr. N. Holbrook (Yale University, New Haven, CT). Probes for Grp78 and GAPDH were prepared from expression plasmids as described previously (24Hong M. Luo S. Baumeister P. Huang J.M. Gogia R.K. Li M. Lee A.S. J. Biol. Chem. 2004; 279: 11354-11363Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The mRNA levels were quantitated by a phosphorimaging device (Molecular Dynamics) using GAPDH as the loading control. The fold of induction was calculated by dividing the relative mRNA level by that of the untreated sample that is set as 1. Western Blot Analysis—The cell lysates from treated or untreated cells were resuspended in lysis buffer (0.5% Nonidet P-40, 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1 mm EDTA, 10% glycerol, 1 mm dithiothreitol, and proteinase inhibitor mixture (1 tablet/10 ml of lysis buffer, Roche Applied Science). After incubation on ice for 20 min, the homogenate was centrifuged at 14,000 revolutions/min for 15 min at 4 °C. Thirty micrograms of total protein of the clarified supernatants was separated by 12 or 14% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed as described previously (23Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). All immunoblots were visualized by ECL (Amersham Biosciences). Immunofluorescence Staining—Cells were plated in 4-well Lab-Tek II glass slides (Nalge Nunc International) a day before treatment and then exposed to TG (2 μm) or Etop (50 μg/ml) for 0, 3, 6, or 24 h. Immunofluorescence staining was performed as previously described (27Meplan C. Mann K. Hainaut P. J. Biol. Chem. 1999; 274: 31663-31670Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Briefly, the treated cells were washed in PBS, fixed in -20 °C 1:1 (v/v) methanol:acetic acid for 5 min, incubated for 1 h in PBS/Nonidet P-40 0.1% containing 5% bovine serum albumin at 4 °C, labeled with the anti-p53 monoclonal antibody Pab241 (1:300 dilution) overnight at 4 °C, and followed by staining with FITC-conjugated horse anti-mouse IgG (1:500 dilution, Vector Laboratories). The cells were mounted in Vectashield with propidium iodide mounting medium (Vector Laboratories) and visualized with a Zeiss LSM 510 dual photon confocal microscope. Apaf-1 Is an Integral Component of ER Stress-induced Apoptosis— Apaf-1 binds to cytochrome c released from the mitochondria and recruits pro-C-9 to form the apoptosome and subsequently activates C-9 and downstream effector caspases (C-3, C-7). Thus, the activation of Apaf-1 serves as a major indicator for mitochondria-mediated apoptosis. To investigate the requirement of the mitochondrial apoptotic pathways, especially Apaf-1, in ER stress-induced apoptosis, Apaf-1-/- and matched WT (Apaf-1+/+) MEFs were either untreated or treated with ER stress inducers TG, Tun, and BFA. Following 24 h of ER stress treatment, the percentage of apoptotic cells was determined by flow cytometry after staining with annexin-V and propidium iodide. For comparison, the cells were also treated with Etop, a DNA-damaging agent known to initiate apoptosis through the mitochondrial pathway (28Perkins C.L. Fang G. Kim C.N. Bhalla K.N. Cancer Res. 2000; 60: 1645-1653PubMed Google Scholar). As shown in Fig. 1A, there was substantial reduction in the percentage of apoptotic cells in Apaf-1-/- MEFs under all treatment conditions, as compared with WT control. Although these results suggest that Apaf-1-mediated apoptotic pathways contribute significantly to cell death during the early phase (within 24 h) of ER stress treatment, it remains possible that, with more severe ER stress treatment and for a longer period of treatment, Apaf-1 could be dispensable. To test this, the same set of cells was treated with higher doses of TG, Tun, and BFA. The kinetics of apoptosis was determined by flow cytometry and cell cycle analysis following PI staining during the treatment period of up to 40 h. Our results reveal that, even under more severe ER stress conditions for prolonged periods of time, MEFs deficient in Apaf-1 were more resistant to apoptosis (20–40% apoptotic cells) as compared with WT cells (>80%) for all three ER stress inducers (Fig. 1B). The same was observed for Etop-treated cells. These observations provide direct evidence that Apaf-1 is an integral part of ER stress-induced cell death in vivo. On the other hand, it is also evident that the inhibition of apoptosis in Apaf-1-/- MEFs is not complete, indicating that pathways independent of Apaf-1 are in operation to execute ER stress-induced apoptosis. ER Stress Activates Caspases through Apaf-1-dependent and -independent Pathways—To examine the effect of Apaf-1 deficiency on caspase activation following ER stress, cell lysates were prepared from Apaf-1-/- and their matched WT MEFs treated with ER stress inducers (TG, Tun, and BFA) and Etop, under conditions as described for Fig. 1A and subjected to Western blot analysis. As shown in Fig. 2, for WT cells, C-7 was activated by TG, Tun, and BFA, as indicated by the detection of the cleaved form of C-7. For C-3, its cleaved form was observed in cells treated with TG but was below the detection limit in cells treated with Tun or BFA. As a control, Etop strongly activated both C-3 and C-7. In Apaf-1-/- MEFs, the activation of both C-3 and C-7 was largely inhibited (Fig. 2). In contrast, C-12 activation by TG and BFA, as evidenced by the appearance of its cleaved form, was independent of Apaf-1 (Fig. 2). Interestingly, in MEFs, Tun only weakly activated C-12, and its cleavage was detected after Etop treatment. The effectiveness of the ER stress inducers was confirmed by their ability to increase the level of the ER chaperone protein GRP78/BiP, and the loading of the protein samples were monitored by β-actin levels. These results indicate that ER stress strongly activates C-7 primarily by mitochondrial pathways downstream of Apaf-1, whereas C-12 activation by ER stress, such as with TG and BFA, is Apaf-1-independent and may contribute to Apaf-1-independent apoptosis in murine MEF cells. The BH3-only Proteins PUMA and NOXA Are Selectively Activated at the Transcript Level upon ER Stress—BH3-only BCL-2 family proteins are regulators of BAK/BAX; as such, they are initiators for the mitochondrial apoptotic pathway. Among the mammalian BH3-only proteins, the transcript levels of BimL, Dp-5, Bnip-3, Nix, Puma, and Noxa could be induced by a variety of stimuli. Their mRNA levels, as well as those encoding Bik and Spike, were measured in primary MEFs following treatment with TG, Tun, and BFA by Northern blot. The induction of Chop served as a positive control, and mRNA loading was monitored by GAPDH levels. Examples of the Northern blots are shown in Fig. 3, A and B, and the relative mRNA levels after TG treatment were quantitated, normalized against the loading control, and summarized in Fig. 3C. Our results show that Bnip-3, Spike, Nix, Bik, and BimL transcript levels were either not affected by ER stress or that the effect was transient and marginal. The level of Dp-5 mRNA was undetectable in MEFs (data not shown). In contrast, Puma and Noxa mRNA levels increased by ∼3–4-fold within 2–4 h under all three ER stress treatment conditions and persisted for at least 16 h. The doublet appearance of the Puma transcripts is consistent with alternative splicing as previously reported (22Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1839) Google Scholar, 29Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar). As a positive control, their mRNA levels were also strongly activated in MEFs treated with Etop (Fig. 3B). Puma and Noxa are target genes of p53 and are critical mediators of the apoptotic responses induced by p53. To test whether the ER stress induction of Puma and Noxa is p53-dependent, mRNA levels of Puma and Noxa were measured in p53-/- and matched WT (p53+/+) MEFs treated with either ER stress inducers (TG, Tun, BFA) or Etop as a positive control. ER stress induction was monitored by Chop mRNA activation and mRNA loading by GAPDH levels. Examples of the Northern blots are shown in Fig. 4A, and the results are summarized in Fig. 4B. We observed that, in primary MEFs, induction of Noxa by all three ER stress inducers was highly dependent on p53, as the induction level was suppressed drastically in p53-/- MEFs. For Puma, mRNA induction by TG, Tun, and BFA was also dependent on p53, although a low level of residual activation could be detected at 4 h. As a positive control, Etop induction of Noxa and Puma transcripts were blocked in p53-/- MEFs. These results indicate that, in MEFs, both Puma and Noxa mRNA induction by ER stress is largely p53-dependent. Suppression of ER Stress-induced Apoptosis in p53-/- MEFs—As induction of Puma and Noxa by ER stress is largely dependent on p53 in MEFs and p53 itself has recently been shown to have a direct signaling role at the mitochondria in the induction of apoptosis (30Leu J.I. Dumont P. Hafey M. Murphy M.E. George D.L. Nat. Cell Biol. 2004; 6: 443-450Crossref PubMed Scopus (641) Google Scholar), we investigated the requirement of p53 in ER stress-induced apoptosis by utilizing the matched pair of WT (p53+/+) and p53-/- MEFs. Etop treatment was used as a positive control. As shown in Fig. 5A, apoptosis induced by TG treatment for 24 h was inhibited by ∼50% in p53-/- MEFs compared with WT cells. This result was confirmed when the kinetics of apoptotic cell death were monitored during a 40-h period (Fig. 5B). These results reveal p53 as a novel component of the ER stress-induced apoptotic signaling pathway in MEFs. Nonetheless, the suppression of apoptosis in the p53 null cells was only partial, indicating that other independent pathways are involved in ER stress-induced apoptosis. ER Stress Induces p53 Level in MEFs and Activates CHOP and C-12 Independent of p53—Because ER stress-induced apoptosis was inhibited in p53-/- MEFs, we examined whether p53 itself undergoes ER stress-induced changes. In WT (Puma+/+) MEFs, by 16 h of TG stress treatment, increase in p53 protein was evident (Fig. 6A). As a positive control, the level of CHOP was elevated by ER s