Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of ischemic and neurodegenerative disorders. Treatment of human SH-SY5Y neuroblastoma cells with tunicamycin, an inhibitor of protein glycosylation, rapidly induced the expression of target genes of the unfolded protein response. However, prolonged treatment also triggered a delayed, caspase-dependent cell death. Microarray analysis of gene expression changes during tunicamycin-induced apoptosis revealed that the Bcl-2 homology domain 3-only family member, Bcl-2 binding component 3/p53 upregulated modulator of apoptosis (Bbc3/PUMA), was the most strongly induced pro-apoptotic gene. Expression of Bbc3/PUMA correlated with a Bcl-xL–sensitive release of cytochrome c and the activation of caspase-9 and -3. Increased expression of Bbc3/PUMA was also observed in p53-deficient human cells, in response to the ER stressor thapsigargin, and in rat hippocampal neurons after transient forebrain ischemia. Overexpression of Bbc3/PUMA was sufficient to trigger apoptosis in SH-SY5Y neuroblastoma cells, and human cells deficient in Bbc3/PUMA showed dramatically reduced apoptosis in response to ER stress. Our data suggest that the transcriptional induction of Bbc3/PUMA may be sufficient and necessary for ER stress–induced apoptosis.
An imbalance between the cellular demand for protein synthesis and the capacity of the ER in promoting protein maturation and transport can lead to an accumulation of unfolded or malfolded proteins in the ER lumen. This condition has been designated “ER stress” (Kaufman, 1999). It has been suggested that ER stress contributes to neuronal injury after cerebral ischemia (Paschen and Frandsen, 2001; DeGracia et al., 2002) and may play an important role in the pathogenesis of neurodegenerative disorders such as Parkinson's and Alzheimer's disease (Sherman and Goldberg, 2001).
ER stress provokes a controlled transcriptional response, the unfolded protein response (UPR). Target genes of the UPR include molecular chaperones that help to alleviate ER stress by promoting the correct folding of nascent proteins in the ER and by inhibiting the aggregation of aberrant proteins. However, if ER stress is severe and prolonged, stressed cells will eventually activate an evolutionary conserved, caspase-dependent death program (Perez-Sala and Mollinedo, 1995). In murine cells, ER stress–induced cell death has been shown to involve the activation of ER-resident caspase-12, which subsequently activates executioner caspases such as caspase-3 (Nakagawa and Yuan, 2000; Nakagawa et al., 2000). However, although the role of caspase-12 in ER stress–induced apoptosis of murine cells is well established, there is also evidence that the human caspase-12 gene is nonfunctional due to the acquisition of inactivating gene mutations (Fischer et al., 2002). This suggests that additional pathways may be important in humans.
Indeed, evidence has been presented that ER stress may activate the mitochondrial apoptosis pathway (Häcki et al., 2000; Annis et al., 2001; Wei et al., 2001). In this pathway, the release of pro-apoptotic factors from mitochondria requires an increase in outer mitochondrial membrane permeability, triggered by the pro-apoptotic Bcl-2 family members Bax and Bak (Desagher and Martinou, 2000; Wei et al., 2001). Both proteins have to undergo a conformational change to trigger this increase. In healthy cells, anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL inhibit this conformational change. On induction of apoptosis, this inhibition is overcome by the transcriptional induction or posttranslational activation of Bcl-2 homology domain 3 (BH3)-only proteins (Huang and Strasser, 2000).
In the present work, we used high density oligonucleotide microarrays to analyze gene expression during tunicamycin-induced ER stress in human neural cells, and provide evidence that the transcriptional induction of the BH3-only protein Bcl-2 binding component 3/p53 upregulated modulator of apoptosis (Bbc3/PUMA) activates the mitochondrial apoptosis pathway in response to ER stress.
Tunicamycin induces caspase-dependent apoptosis in neurons
Treatment of human SH-SY5Y neuroblastoma cells with the ER stressor tunicamycin induced mRNA and protein expression of the ER resident molecular chaperone immunoglobulin heavy chain–binding protein (BiP), a known UPR target gene (Fig. 1, A and B). Despite this protective response, prolonged treatment with tunicamycin resulted in apoptotic cell death. FACS® analysis of the DNA content demonstrated that tunicamycin treatment induced a characteristic sub-G1 peak in the human neuroblastoma cells, indicative of DNA fragmentation, a hallmark of apoptosis (Fig. 1 C). After 24 h, ∼50% of cells had a reduced DNA content (Fig. 1 D, sub-G1). Co-incubation with the broad-spectrum caspase inhibitor zVAD-fmk blocked the appearance of sub-G1 cells, indicating that this process was caspase-dependent. Indeed, tunicamycin induced a caspase-3–like protease activity in cytosolic extracts determined by measuring the cleavage of Ac-DEVD-AMC, a fluorogenic substrate preferentially cleaved by caspase-3 and -7 (Fig. 1 E). Treatment with tunicamycin also induced apoptosis in primary cultures of rat hippocampal neurons (Fig. 1 F) reaching a maximum at a concentration of 10–30 μM (Fig. 1 G). Treatment with tunicamycin also led to an early induction of the molecular chaperone BiP in the hippocampal neuron cultures (unpublished data).
Gene expression microarray analysis of tunicamycin-treated SH-SY5Y cells
We used high density oligonucleotide microarrays to analyze gene expression changes during tunicamycin-induced cell death. RNA was isolated from SH-SY5Y neuroblastoma cells treated for 4, 8, or 12 h with 3 μM tunicamycin. A total of 138, 225, and 151 genes were found to be up-regulated after 4, 8, and 12 h of tunicamycin treatment, respectively (see Materials and methods for detailed description of data analysis). The number of down-regulated genes was 100, 69, and 88 after 4, 8, and 12 h, respectively. Table I provides a list of selected, differentially expressed genes after ER stress, sorted into distinct functional subgroups. Column 4 refers to the expression kinetics shown in Fig. 2 A. Expression kinetics were classified into persistent, early, intermediate, or late changes (Fig. 2 A). Increased expression of distinct ER stress–induced genes was confirmed by semi-quantitative RT-PCR analysis (Fig. 2 B). A list of all differentially expressed genes is provided in the supplemental material (available).
Genes involved in protein folding
The largest group of up-regulated target genes was represented by ER-resident molecular chaperones and disulfide isomerases. Among the molecular chaperones, we found established UPR target genes such as BiP, 94-kD glucose-regulated protein, oxygen-regulated protein 150 (ORP150), and the fourth mammalian ER DNAJ protein (ERdj4). Other activated genes included disulfide isomerases such as protein disulfide isomerase ER-60 precursor (ERp60) and the proline 4-hydroxylase, β subunit (P4HB). Protein folding is catalyzed and accelerated by members of the peptidyl prolyl cis/trans isomerase family of which cyclophilin B and FK506-binding protein 13 (FKBP13) were potently up-regulated by tunicamycin. Interestingly, the expression of the chaperone 71-kD heat shock cognate protein (hsc70) was significantly lowered during ER stress. Other chaperones down-regulated on tunicamycin treatment included DNAJ-like 2, the 90-kD heat shock protein, and the 28-kD heat shock protein.
Genes involved in protein transport
The second major group of target genes included genes involved in cotranslational translocation of proteins across the ER membrane and the anterograde/retrograde transport back into the cytosol. This group included the signal recognition particle receptors SRP54 and SRP19, the signal sequence receptors SSR1 and 4, members of the SEC61 complex (SEC61 β and γ, as well as SEC63), translocating chain–associating membrane protein (TRAM) and translocon-associated protein delta subunit (TRAP δ). Up-regulated genes also included genes involved in the anterograde/retrograde transport between ER and Golgi. These were represented by a coated vesicle membrane protein (rnp24), a transmembrane trafficking protein (tmp21), and syntaxin 5. Other up-regulated genes involved in intracellular protein trafficking included the mitochondrial inner membrane translocase hTIM44.
Genes involved in protein synthesis
The translational machinery for a new boost of protein synthesis has been suggested to be amplified during UPR-associated translational attenuation (Okada et al., 2002). We observed an up-regulation of eight tRNA synthetase genes after treatment with tunicamycin. Asparagine synthetase, an activating transcription factor 4 (ATF4) target gene, was also significantly induced.
Genes regulating Ca2+ homeostasis
Members of this group included calreticulin and calnexin. Both proteins regulate ER Ca2+ homeostasis, but also function as Ca2+-dependent ER chaperones. Genes involved in the regulation of Golgi Ca2+ homeostasis included nucleobindin 1 and novel DNA binding/EF-hand/leucine zipper protein (NEFA). Of note, two members of the P-type transport ATPase family, the plasma membrane Ca2+ pumping ATPase (ATP2B1) and sarcoplasmic reticulum Ca2+-ATPase 2 (ATP2A2) were also induced.
Up-regulated transcription factors included two established UPR target genes: C/EBP homologous protein (CHOP) and X box–binding protein 1 (XBP-1). Both transcription factors were persistently activated with a relatively even expression level at all three time points investigated. CHOP showed the largest expression increase of all genes analyzed. Interestingly, expression of the stress-responsive transcriptional regulator early growth response 1 (Egr-1) was also significantly enhanced. In addition, the CHOP interaction partner CCAAT/enhancer binding protein beta (C/EBP-β) was potently induced.
ER stress induced by tunicamycin activates the BH3-only gene bbc3/PUMA
Tunicamycin-induced ER stress also altered the transcription of bbc3/PUMA, a member of the BH3-only gene family. Microarray analysis could not detect an altered transcription of other Bcl-2 family members, including the BH-3-only genes bim, bad, and ATL-derived PMA-responsive peptide (APR; human Noxa), the pro-apoptotic Bcl-2 family members bax and bak, and the anti-apoptotic genes bcl-2 and bcl-x. There was a mild induction of DR5/Trail-R2 (2.1-fold increase after 8 h), but the expression of other genes involved in death receptor–mediated apoptosis including Fas/CD95/APO-1, Fas ligand, TRAIL, and TRAIL-Receptor DR3, was not found to be altered.
Northern blot analysis demonstrated that up-regulation of bbc3/PUMA expression could be observed 4 h after addition of tunicamycin (Fig. 3 A). Cell lysates of SH-SY5Y cells treated with 3 μM tunicamycin showed elevated Bbc3/PUMA protein levels starting 12 h after onset of treatment (Fig. 3 B, top). Parallel analysis of caspase-3 activation revealed an ensuing appearance of active caspase-3 subunits detected with an antibody predominantly recognizing the p20 subunit (Fig. 3 B, middle). In concordance with the microarray data, we failed to detect significant protein induction of Bim and Bid (Fig. 3 C), two BH3-only proteins previously implicated in stress-induced apoptosis (Bouillet et al., 1999; Plesnila et al., 2001; Putcha et al., 2001; Whitfield et al., 2001).
Induction of Bbc3/PUMA is a general, p53-independent cellular response to prolonged ER stress
bbc3/PUMA has been described as a p53 target gene (Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001). However, the microarray data did not reveal significant induction of classical p53 target genes such as p21 and human mdm2. Although Western blot analysis demonstrated significant p53 accumulation and p21 protein induction in SH-SY5Y cells treated with the topoisomerase II inhibitor etoposide, tunicamycin treatment had no effect on p53 and p21 protein levels (Fig. 4 A). Moreover, tunicamycin increased bbc3/PUMA mRNA and protein levels in p53-deficient human SAOS-2 osteosarcoma cells (Fig. 4, B and C).
bbc3/PUMA mRNA (Fig. 5 A) and protein (unpublished data) levels also increased in SH-SY5Y neuroblastoma cells exposed to the ER Ca2+ ATPase inhibitor thapsigargin, demonstrating that the activation of bbc3/PUMA was observed after treatment with a second, pharmacologically distinct ER stressor. Tunicamycin also increased the expression of Bbc3/PUMA in cultured rat hippocampal neurons (Fig. 5 B), demonstrating that the activation of bbc3/PUMA was not restricted to transformed human cells.
Cerebral ischemia is a pathophysiological ER stressor (Paschen and Frandsen, 2001; DeGracia et al., 2002). In male Wistar rats subjected to 10 min of forebrain ischemia, increased nuclear accumulation of the transcription factor CHOP could be detected in hippocampal pyramidal neurons of the CA1 subfield as early as 1 h after ischemia (Fig. 5 C, top), indicating that ischemia/reperfusion injury induced a rapid ER stress response in selectively vulnerable brain regions. Expression of Bbc3/PUMA was not detectable during the first 24 h after transient forebrain ischemia, but increased expression of CHOP could still be observed (unpublished data). However, expression of Bbc3/PUMA increased significantly in CA1 neurons 48–120 h after ischemia (Fig. 5 C, bottom). This correlated with the onset of neuronal degeneration (Fig. 5 C) and the occurrence of TUNEL-positive nuclei in the CA1 hippocampal pyramidal cells (Rami et al., 2000). Western blot analysis of the hippocampus and neocortex tissue of ischemic and sham-operated animals 72 h after surgery also provided evidence for a significant induction of Bbc3/PUMA expression in the selectively vulnerable hippocampus (Fig. 5 D).
Activation of the mitochondrial apoptotic pathway during tunicamycin-induced apoptosis
Activation of bbc3/PUMA suggested that the mitochondrial apoptosis pathway was activated during ER stress–induced apoptosis. Indeed, treatment with tunicamycin led to Bax activation and clustering in SH-SY5Y neuroblastoma cells, as demonstrated by immunofluorescence analysis using an antibody that specifically detects a conformational change in the Bax protein required for its pro-apoptotic activity (Fig. 6 A). Cells exhibiting Bax clustering showed apoptotic morphology and increased chromatin condensation. Cells with increased chromatin condensation also lost their typical filamentous cytochrome c staining (Fig. 6 B), indicating that cytochrome c was released from mitochondria. Of note, the apoptotic phenotype of SH-SY5Y cells was always associated with markedly enhanced Bbc3/PUMA immunostaining. Tunicamycin treatment also led to activation of caspase-9, the apical caspase in the mitochondrial apoptosis pathway (Fig. 6 C).
Subsequently, we investigated the effect of Bcl-xL overexpression on tunicamycin-induced apoptosis using SH-Neo and SH-Bcl-xL cells (Luetjens et al., 2001). SH-Neo and SH-Bcl-xL cells responded with increased expression of Bbc3/PUMA and BiP to an exposure to 3 μM tunicamycin (Fig. 6 D; unpublished data). Selective digitonization of the plasma membrane and subsequent immunoblotting experiments revealed that tunicamycin induced a significant accumulation of cytochrome c in the cytosol in SH-Neo cells (Fig. 6 E). 3 μM staurosporine (STS) also induced a prominent release of cytochrome c into the cytosol of SH-Neo cells. This release was more pronounced than the release triggered by tunicamycin. However, in direct comparison with tunicamycin, exposure to STS also led to a more rapid and extensive cell death (>80% cell death after 18 h; Luetjens et al., 2001). Interestingly, both tunicamycin- and STS-induced release of cytochrome c release could not be observed in cells overexpressing Bcl-xL. Overexpression of Bcl-xL also potently inhibited the activation of caspase-3–like proteases (DEVDases) in response to tunicamycin and STS (Fig. 6 F).
Expression of bbc3/PUMA may be sufficient and required for ER stress–induced apoptosis in human cells
To test whether expression of bbc3/PUMA was sufficient to kill human SH-SY5Y neuroblastoma cells, cells were cotransfected with a plasmid encoding EGFP (pEGFP) and either hemagglutinin (HA)-tagged Bbc3/PUMA (pHA-Bbc3/PUMA) or empty vector (pcDNA3.1). Hoechst 33258 staining and morphological analysis of GFP-positive cells demonstrated that bbc3/PUMA-transfected cells revealed morphological characteristics of apoptosis, including cell shrinkage and chromatin condensation/fragmentation 16 h after transfection (Fig. 7 A). In contrast, empty vector-transfected controls remained morphologically largely intact. Quantitative FACS® analysis of propidium iodide of EGFP-positive SH-SY5Y neuroblastoma cells transiently cotransfected with expression plasmids encoding either HA-tagged bbc3/PUMA (pHA-bbc3/PUMA), HA-tagged bbc3/PUMA with a deletion in the BH3-domain (pHA-bbc3/PUMA-ΔBH3), or empty vector (pcDNA3.1) demonstrated prominent cell death in bbc3/PUMA-transfected cells, reduced cell death in bbc3/PUMA-ΔBH3–transfected cells (Han et al., 2001), and little cell death in empty vector-transfected cells (Fig. 7 B). Parallel Western blot analysis using an anti-HA antibody showed comparable transfection rates in the pHA-bbc3/PUMA– and pHA-bbc3/PUMA-ΔBH3–transfected cells (Fig. 7 B).
To investigate whether Bbc3/PUMA induction was required for ER stress–induced apoptosis in human cells, we exposed HCT116 control cells and HCT116 cells with targeted disruptions of both alleles of the bbc3/PUMA gene (Yu et al., 2003) to the ER stressor thapsigargin (1 μM). Western blot analysis demonstrated a comparable induction of the UPR target gene product BiP in response to thapsigargin in control and Bbc3/PUMA-deficient cells (Fig. 7 C). HCT116 control cells also showed significant induction of Bbc3/PUMA protein in response to thapsigargin. Interestingly, FACS® analysis of DNA content in HCT116 control and Bbc3/PUMA-deficient cells exposed to thapsigargin demonstrated significantly reduced apoptosis in the Bbc3/PUMA-deficient cells (Fig. 7, D and E), whereas the response to STS was not altered (Fig. 7 E).
ER stress response and protein quality control
In this analysis, we have used high density oligonucleotide microarrays to analyze transcriptional changes during ER stress–induced apoptosis in human SH-SY5Y neuroblastoma cells. Among the early and persistently induced genes, we found genes that relieve ER stress by promoting protein folding and by keeping proteins in a folding-competent state. This group was represented by molecular chaperones of the classical (BiP, GRP94) and nonclassical (lectin-like molecular chaperones like calnexin and calreticulin) type, along with protein disulfide isomerases. Also, we observed the induction of the sarcoplasmic reticulum Ca2+-ATPase 2 ATP2A2 (SERCA2) and the plasma membrane Ca2+ pumping ATPase (ATP2B1). Up-regulation of SERCA2 has previously been observed in response to different ER stressors (Hojmann Larsen et al., 2001). Up-regulation of both plasma membrane- and ER-localized Ca2+-ATPases may help to relieve ER stress by refilling of ER Ca2+ stores.
A distinct group of genes differentially regulated by tunicamycin is involved in cotranslational translocation of proteins across the ER membrane, protein trafficking to other organelles such as mitochondria, and proteins involved in retrograde transport back into the cytosol. With the exception of translocating chain–associating membrane protein, this group of genes was induced late during ER stress. Induction of members of the SEC61 complex may link the UPR with ER-associated degradation (ERAD; Wiertz et al., 1996; Plemper et al., 1997). Hence, ER stress may result in a highly ordered gene regulation process comprised of different sequential steps: an early up-regulation of molecular chaperones in order to keep nonglycosylated proteins in a folding-competent state, followed by an improvement of the capacity of the secretory pathway. If ER stress persists, the ERAD pathway may be switched on to target unfolded proteins to degradation via the proteasome.
Transcriptional regulation of ER stress
The transcription factor CHOP was the gene most strongly induced by tunicamycin. In overexpression studies, CHOP has been shown to negatively regulate cell cycle and induce apoptosis (Barone et al., 1994; Zinszner et al., 1998). The transcription factor C/EBP-β, an interaction partner of CHOP, was also strongly up-regulated during ER stress. CHOP acts as a dominant-negative regulator of C/EBPs and inhibits the DNA-binding activity of C/EBP-β by forming heterodimers that cannot bind DNA (Ron and Habener, 1992). Therefore, CHOP may have a dual function; first acting as an inhibitor of C/EBP proteins to activate distinct target genes, and second, as a direct activator of CHOP target genes (Ubeda et al., 1996). Notably, we could not detect increased expression of known “downstream of CHOP” (doc) genes (Wang et al., 1998) such as homologues of the actin-binding proteins villin and gelsolin and carbonic anhydrase VI (CA-VI). This suggests that CHOP does not function independently of other transcription factors (like C/EBP-β) during ER stress, and favors the idea that transcription is limited to genes activated by heterodimers of CHOP-C/EBP-β. We also observed the potent induction of the transcription factor Egr-1, which was activated early and transiently after ER stress. Egr-1 has been shown to be induced as an “immediate-early gene” during several physiological and pathophysiological stress conditions, including ischemia (Yan et al., 2000). Egr-1 has previously been linked to cell death induced by the ER Ca2+ ATPase inhibitor thapsigargin, and has been shown to induce apoptosis in human melanoma cells (Muthukkumar et al., 1995).
ER stress and apoptosis
There are two possible outcomes of prolonged ER stress: (1) an adaptive response promoting cell survival; or (2) the induction of apoptotic cell death. Among the pro-apoptotic genes, the BH3-only gene bbc3/PUMA was significantly up-regulated after prolonged tunicamycin treatment. Bbc3/PUMA was likewise up-regulated after an exposure to thapsigargin, which induces ER stress by depleting ER Ca2+ stores. bbc3/PUMA has previously been identified as a p53 target gene (Nakano and Vousden, 2001; Yu et al., 2001). However, induction of bbc3/PUMA has also been shown to involve p53-independent regulation (Han et al., 2001). Our results demonstrate that the induction of bbc3/PUMA links the cellular ER stress response to the mitochondrial apoptosis pathway, and that this may occur independently of p53. Of note, human cells deficient in Bbc3/PUMA showed dramatically reduced apoptosis in response to ER stress. Interestingly, they still maintained a very low level of apoptosis (Fig. 7 E), suggesting that the (posttranslational) activation of alternative BH3-only proteins or mitochondria-independent apoptosis pathways may only partially compensate for a Bbc3/PUMA deficiency. Induction of Bbc3/PUMA was also observed in primary neuron cultures and in response to cerebral ischemia in vivo, suggesting that regulation of bbc3/PUMA expression is a common pathway during ER stress–induced cell death in mammals. Our finding that BH3-only family members are also involved in apoptotic signaling originating from the ER emphasizes the importance and diversity of this gene family in cellular life and death decisions.
Materials And Methods
Tunicamycin, thapsigargin, etoposide, and STS were purchased from Qbiogene. Caspase substrate acetyl-DEVD-7-amido-4-methylcoumarin (Ac-DEVD-AMC) and the broad-spectrum caspase inhibitor Z-Val-Ala-Asp(O-methyl)-fluoromethylketone (zVAD-fmk) were obtained from Bachem. Hoechst 33258 was purchased from Sigma-Aldrich.
Cell lines, cell culture, and transfections
Human SH-SY5Y neuroblastoma cell lines were grown in RPMI 1640. The generation of SH-SY5Y neuroblastoma cells stably overexpressing Bcl-xL (SH-Bcl-xL) or stably transfected with the empty vector (SH-neo) has been described previously (Luetjens et al., 2001). Human SAOS-2 osteosarcoma cells completely lacking expression of endogenous p53 were grown in DMEM culture medium. HCT116 control and Bbc3/PUMA knock-out cells (provided by Drs. B. Vogelstein, L. Zhang, and J. Yu, Johns Hopkins Oncology Center, Baltimore, MD) were maintained in McCoy's 5A medium. All media were supplemented with 10% (vol/vol) FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Primary cultures of hippocampal neurons were prepared from neonatal postnatal day 1 (P1) Fischer 344 rats as described previously (Luetjens et al., 2001). Experiments were performed on 10- to 14-d-old cultures. Animal care followed official governmental guidelines.
For transient transfections, SH-SY5Y cells were plated onto 6-well plates (Nunc). 1 d later, cells were transfected with plasmids encoding HA-tagged, full-length bbc3/PUMA expression vector (pHA-bbc3/PUMA), HA-tagged, bbc3/PUMA expression vector with a deletion of the BH3 domain (pHA-bbc3/PUMA-ΔBH3; provided by Dr. B. Vogelstein), an expression vector for eGFP (pEGFP-C1; CLONTECH Laboratories, Inc.), or empty vector (pcDNA3.1; Invitrogen) using LipofectAMINE™ transfection reagent (Invitrogen). Transfection procedure was followed as described before (Poppe et al., 2002).
SH-SY5Y neuroblastoma cells were treated with tunicamycin or vehicle (0.1% DMSO), harvested, and total RNA was extracted using the RNeasy Midi Kit (QIAGEN). cDNA was synthesized from 40 μg of the total RNA by using the SuperScript™ Choice Kit (Invitrogen). The cRNA was prepared and biotin labeled by in vitro transcription (Enzo Biochemical). Labeled cRNA was fragmented and hybridized for 16 h at 45°C to an HG-U95A array (Affymetrix, Inc.). After hybridization, the gene chips were automatically washed and stained with streptavidin-phycoerythrin by using a fluidics station (GeneChip Fluidics Station 400; Affymetrix, Inc.). Finally, probe arrays were scanned at a 3-μm resolution using the Genechip System confocal scanner (Aligent Technologies). Microarray Suite 4.1 (Affymetrix, Inc.) was used to scan and analyze the relative abundance of each gene from the average difference of intensities. Analysis parameters used by the software were set to values recommended by Affymetrix, Inc (SDT = 4.0, SRT = 1.5). All chip data were normalized to a target intensity of 1000. For each individual gene, the signal ratio between perfect match and mismatch probe cells was used to determine its “Absolute Call,” indicating whether the corresponding transcript was present (P), absent (A), or marginal (M). For analysis of differential target gene expression, the “Difference Call Decision Matrix” was used. Individual transcript levels were ranked as either increased (I), marginally increased (MI), decreased (D), marginally decreased (MD), or not changed (NC). To be considered as differentially regulated, the Difference Call of individual transcripts had to be increased/decreased or marginally increased/marginally decreased at the respective time point after treatment with tunicamycin, whereas the Absolute Call had to be present or marginal at the respective time point (up-regulated genes) or in the control (down-regulated genes). A list of all differentially regulated genes meeting this criteria is provided in the online supplemental materials (available). Genes were also classified according to their expression kinetics. The highest fold-induction in comparison to control was set to 100%. Genes up- and down-regulated to at least 50% for all three time points were considered to be persistently regulated. In all other cases, genes were ranked as early (4 h), intermediate (8 h), or late (12 h), depending on the time point when the maximal change of the corresponding transcript was reached. The output from the microarray analysis was merged with the Unigene or GenBank descriptor and stored as an Excel (Microsoft) data spreadsheet. For confirmation of gene chip data, a replication experiment with a second set of independent samples was performed, revealing similar results.
The release of cytochrome c from mitochondria was analyzed by a modified selective digitonin permeabilization method that was described previously (Wobser et al., 2002).
SDS-PAGE and Western blotting
Preparation of cell lysates, Western blotting, and immunodetection was performed as described previously (Poppe et al., 2002). The blots were incubated with a mouse monoclonal anti-BiP antibody (SPA 827; StressGen Biotechnologies) diluted 1:1,000, a mouse monoclonal anti-Bbc3 antibody (KM140; Han et al., 2001) diluted 1:1,000, a rat monoclonal Bim antibody (clone 14A8; Qbiogene) diluted 1:1,000, a rabbit polyclonal Bid antibody (#2315-PC; Trevigen) diluted 1:1,000, a rabbit polyclonal p53 antibody (NCL-p53-CM1; Loxo) diluted 1:1,000, a mouse monoclonal p21/WAF antibody (Ab-4, #OP 76; Oncogene Research Products) diluted 1:500, a mouse monoclonal cytochrome c antibody (clone 7H8.2C12; BD Biosciences) diluted 1:1,000, a rabbit polyclonal anti-caspase-3 antibody (H-277; Santa Cruz Biotechnology, Inc.) diluted 1:1,000, a rabbit polyclonal anti-caspase-9 antibody (#556585; BD Biosciences) diluted 1:1,000, a mouse monoclonal anti-hemagglutinin (HA) antibody (12CA5; Roche) diluted 1:1,000, or a mouse monoclonal anti-α-tubulin antibody (DM 1A; Sigma-Aldrich), diluted 1:20,000.
Validation of up-regulated target gene expression was performed by RT-PCR analysis for selected genes. We designed unique oligonucleotide primer pairs by using PRIMER3 software (www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Primer sequences are available on request from the authors. Total RNA, purified from tunicamycin-treated cells using the RNeasy Mini kit (QIAGEN), was subjected to RT-PCR in a two-step protocol using M-MLV reverse transcriptase (Invitrogen) and Taq polymerase (Eppendorf; Kögel et al., 2003). The number of cycles and annealing temperature was adjusted depending on the genes amplified.
Northern blot analysis
Total RNA was isolated using the RNeasy Mini or Midi Kit (QIAGEN). 10 μg RNA was subjected to gel electrophoresis on 1.1% formaldehyde/1.2% agarose gels and transferred to nylon membranes (Amersham Biosciences). After UV cross-linking and prehybridization in 50% formamide, 5× SSC, 5× Denhardt's solution, and 1% SDS, pH 7.4, at 65°C for 30 min, the blots were hybridized with an α[32P]dCTP-labeled full-length human bbc3/PUMA cDNA probe (Han et al., 2001) and a 700-bp fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 50% formamide, 5× SSC, 5× Denhardt's solution, and 1% SDS, pH 7.4, at 47°C, and were washed according to standard procedures. Membranes were exposed to X-ray film for 24 to 72 h.
Flow cytometry analysis
The measurement of propidium iodide uptake and DNA leakage from apoptotic nuclei and was performed as described previously (Poppe et al., 2002).
Determination of caspase-3–like protease activity
Caspase-3–like protease activity of cells exposed to tunicamycin or vehicle was determined as described before (Poppe et al., 2002).
Transient forebrain ischemia
Transient forebrain ischemia was performed in male Wistar rats (250–300 g; Smith et al., 1984). Cerebral ischemia was induced by clamping both common carotid arteries and lowering the mean arterial blood pressure to 40 mm Hg. After 10 min ischemia, the blood pressure was restored by re-infusion of blood and removal of the clamps. Arterial pH, pCO2, pO2, arterial blood pressure, and plasma glucose concentration were determined 15 min before and 15 min after the induction of ischemia. Sham-operated rats served as controls. Animal care followed official governmental guidelines.
At different time points after cerebral ischemia, rats were anaesthetized with 3.5% halothane and perfused transcardially with phosphate buffer (pH 7.4) containing 4% formaldehyde. The sections were deparaffinized, washed, and subjected to histochemical analysis as described previously (Rami et al., 2000). Sections were incubated with an mAb against CHOP (sc-7351, Santa Cruz Biotechnology, Inc.) diluted 1:10, or a pAb against Bbc3/PUMA (provided by Dr. Karen Vousden (Beatson Institute for Cancer Research, Glasgow, UK; Nakano and Vousden, 2001) diluted 1:50 overnight at 4°C. The immunoreaction was developed with 3,3′-DAB tetrahydrochloride and hydrogen peroxide. Sections were counterstained with cresyl violet.
Immunofluorescence analysis and Hoechst staining
Tunicamycin-treated cells were washed, fixed with 4% PFA, and permeabilized. A rabbit polyclonal anti-Bax antibody specifically recognizing active Bax (Desagher et al., 1999; #06–499, Upstate Biotechnology) diluted 1:500, the KM140 mouse monoclonal antibody specific for Bbc3 diluted 1:50, or a mouse monoclonal anti-cytochrome c antibody (clone 6H2.B4, #65971; BD Biosciences) diluted 1:1,000 were used to detect the respective proteins. After washing, biotin-conjugated anti–mouse or anti–rabbit IgG (1:1,000; Vector Laboratories) was added for 1 h, followed by a 20 min treatment with streptavidin-Oregon green conjugate (1 μg/ml; Molecular Probes, Inc.). In case of the cytochrome c staining, a Texas red–coupled anti–mouse antibody was used (T-0300; Molecular Probes, Inc.). As a negative control, cultures were incubated with the secondary antibody only. Fluorescence was observed using an inverted microscope (Eclipse TE300; Nikon). Digital images of equal exposure were acquired with the SPOT-2 camera using SPOT software version 2.2.1 (Diagnostic Instruments). Nuclei were stained with Hoechst 33258 (1 μg/ml in PBS) for 20 min.
Data are given as means ± SEM. For statistical comparison, t test or one-way ANOVA followed by Tukey test were used. P values smaller than 0.05 were considered to be statistically significant.
Online supplemental material
Supplemental material lists 711 genes that showed differential expression during tunicamycin-induced ER stress analyzed using Affymetrix, Inc. U95A microarrays and Microarray Suite 4.1 software. For each gene, the probe set number, the absolute call (P, present; M, marginal), difference call (D, decrease; I, increase; MD, marginal decrease; MI, marginal increase), the fold change value, and the description containing the accession no. are shown.
The authors thank Hanni Bähler, Christiane Schettler, Hildegard Schweers, Petra Mech, and Wilfried C. Magura for excellent technical assistance; Drs. B. Vogelstein, L. Zhang, and J. Yu for Bbc3/PUMA-deficient cells and reagents; and Dr. K. Vousden for antiserum.
This work was supported by grants from Alzheimer Forschung Initiative e.V. (00808), Deutsche Forschungsgemeinschaft (PR 338/9-3), and the Interdisciplinary Center for Clinical Research, University Münster Clinics (BMBF grant 01 KS 9604/0) to J.H.M. Prehn.
The online version of this article includes supplemental material.
Abbreviations used in this paper: Bbc3/PUMA, Bcl-2 binding component 3/p53 upregulated modulator of apoptosis; BiP, immunoglobulin heavy chain–binding protein; CHOP, C/EBP-homologous protein; Egr-1, early growth response 1; HA, hemagglutinin; STS, staurosporine; UPR, unfolded protein response.