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The Journal of Immunology, 2006, 176: 6245-6253.
Copyright © 2006 by The American Association of Immunologists

C/EBP Homologous Protein (CHOP) Is Crucial for the Induction of Caspase-11 and the Pathogenesis of Lipopolysaccharide-Induced Inflammatio1

Motoyoshi Endo*, Masataka Mori*, Shizuo Akira{dagger} and Tomomi Gotoh2,*

* Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; and {dagger} Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 is a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-mediated apoptosis. To determine whether the ER stress-CHOP pathway is involved in the pathogenesis of the lung inflammation, mice were given LPS intratracheally. Treatment with LPS induced mRNAs for CHOP and BiP. The LPS-induced inflammation in lung, including the IL-1beta activity in bronchoalveolar lavage fluid, was attenuated in the Chop knockout mice. Caspase-11, which is needed for the activation of procaspase-1 and pro-IL-1beta, was induced by LPS treatment in the lung and primary cultured macrophages. The induction of caspase-11 by LPS was suppressed in Chop knockout mice. Caspase-11 was also induced by such ER stress inducers as thapsigargin or tunicamycin. These results show that CHOP plays a crucial role in the pathogenesis of inflammation through the induction of caspase-11.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The endoplasmic reticulum (ER)3 performs several important functions, including posttranslational modification, folding, and the assembly of newly synthesized secretory and cell membrane proteins, and its proper function is essential to cell survival (1). When the cells are subjected to ER stress due to the accumulation of unfolded proteins in the ER, the ER stress pathways are activated to protect the cells (2). These responses involve induction of ER chaperones including Ig H chain binding protein (BiP), translational attenuation, and the degradation of unfolded proteins by a system called ER-associated degradation. Recently, the ER stress pathways have been reported to also be activated by various stresses such as oxidative stress, hypoxia, ischemia, and poly Q proteins (3, 4, 5). Therefore, these pathways function as an intracellular stress sensor and protect cells from various stresses. However, when the ER functions are severely impaired, then apoptosis occurs (6). This apoptosis is mediated by C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 or other factors (7). CHOP is a member of the C/EBP transcription factor family, is induced by ER stress and thus causes apoptosis. However, the target gene(s) of CHOP are still not clear at present. We found that a proapoptotic Bcl-2 family molecule Bax is translocated from cytosol to mitochondria thereby transducing the apoptosis signal in ER stress- and CHOP-mediated apoptosis (8). We also found that ER stress- and CHOP-mediated apoptosis plays a key role in mouse hereditary diabetes mellitus caused by insulin gene mutation (9), NO-induced apoptosis in pancreatic beta cells (10), microglias (11), and macrophages (12), neuronal cell death induced by ischemia-reperfusion (4), and nonsteroidal anti-inflammatory drug-induced apoptosis in gastric mucosal cells (13). We speculate that CHOP-mediated apoptosis may thus be involved in many other diseases.

Severe pneumonia remains one of the major clinical problems, and the mortality rate is high. Various cytokines, secreted from macrophages and other inflammatory cells, are involved in the pathogenesis of pneumonia and many other inflammatory diseases (14). IL-1beta is secreted from activated macrophages at an early stage of the inflammatory response and it also activates other inflammatory cells including the macrophage themselves (15). In activated macrophages, IL-1beta is synthesized as pro-IL-1beta and it is then processed into mature IL-1beta by caspase-1 (16). Actually, the activation of IL-1beta was suppressed in LPS-treated caspase-1 knockout mice (17), and these mice were resistant to the lethal effects of LPS. Caspase-1 is also known as IL-1beta-converting enzyme (ICE). However, caspase-1 (mRNA) is also expressed in non-IL-1beta-secreting cells. Therefore, it has been speculated that the function of caspase-1 is not limited to ICE. Miura et al. (18) showed that an overexpression of caspase-1 gene in a cultured cell line caused the cells to undergo apoptosis. Caspase-1 is also synthesized as procaspase-1, and it exists in cytosol as a proform in nonactivated cells. In activated macrophages, procaspase-11 is induced and activated by autoproteolysis. The active form of caspase-11 activates caspase-1 by proteolysis. Wang et al. (19) showed that caspase-11 knockout mice are resistant to endotoxin shock induced by LPS. They also showed that the expression of caspase-11 is essential for the activation of caspase-1. Both caspase-1 and caspase-11 belong to the inflammatory caspase subfamily. Caspase-11 is induced through the pathway involving NF-{kappa}B and STAT1 in LPS-treated macrophages (20).

In this study, we report that the CHOP-involved pathway plays a crucial role in both the induction of caspase-11 and in the pathogenesis of LPS-induced lung injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Polyclonal Abs against CHOP, BiP, caspase-1, and mAb against heat shock cognate (Hsc) 70 were obtained from Santa Cruz Biotechnology. mAb against caspase-11 was obtained from Sigma-Aldrich.

Generation of animal models

All procedures were approved by the Animal Care and Use Committee of Kumamoto University. Mice lacking the Chop gene (C57BL/6 background) were generated as previously described (10). The tracheae of specific pathogen-free male C57BL/6 and Chop knockout mice weighing 19–25 g (6 wk of age) were cut-down and given Escherichia coli LPS (serotype 0127:B8; Sigma-Aldrich) intratracheally at 10 mg/kg body weight (total volume was set to 30 µl/body), or recombinant mouse IL-1beta (Genzyme-Techne) intratracheally at 5 mg/kg body weight (total volume was set to 30 µl/body) and then killed at the indicated times after being anesthetized with ether.

Bronchoalveolar lavage (BAL), the measurement of cytokines in BAL fluid (BALF), and the staining of alveolar cells

Mice lungs were lavaged intratracheally with PBS (pH 7.4, 0.7 ml) twice, and then BALF was obtained (~1.0 ml). To obtain samples for the measurement of cytokines, BALF samples were centrifuged at 820 x g for 5 min at 4°C, and then the supernatants were used for ELISA to measure the concentration of TNF-{alpha} and IL-1beta. ELISA for TNF-{alpha} and IL-1beta were performed using mice TNF-{alpha} and IL-1beta ELISA kit (BioSource International) according to the protocol provided by the manufacturer. To obtain alveolar cells, BALF were applied to cytospin slides and then underwent centrifugation at 820 x g for 5 min at 4°C. The staining of alveolar cells was performed using Diff-Quik (Sysmex) according to the protocol provided by the manufacturer and then were observed by microscopy. The percentages of each kind of alveolar cell were calculated morphologically. More than 500 cells in each condition were analyzed.

Isolation and treatment of peritoneal macrophages

Mice peritoneal macrophages were prepared and grown in RPMI 1640 medium supplemented with 10% FCS as described (21). After being cultured for 3 days, nonadherent cells were removed, and then adherent cells were treated with LPS (150 µg/ml) or thapsigargin (4 µM) or tunicamycin (1 µg/ml) for the indicated periods.

H&E staining

The lungs were fixed by perfusing with 4% paraformaldehyde in PBS (pH 7.4), embedded in paraffin, and sectioned 5-µm thick. The procedure for H&E staining has been previously described (22).

RT-PCR analysis

The mice were killed with deep anesthetization at the indicated times after treatment. The lungs were removed and frozen immediately on dry ice then stored at –80°C. Total RNA from lung and cultured cells were isolated using the acid guanidium thiocyanate phenol-chloroform extraction procedure as described (23). cDNA was synthesized using the Superscript One-Step RT-PCR System (Invitrogen Life Technologies). The primers used for PCR are as follows: sense primer, 5'-ACTCATTGTGGCTGTGGAGAAG-3' and antisense primer, 5'-GCCGTCTTTCATTACACAGGAC-3' for IL-1beta (GenBank accession number M15131); sense primer, 5'-TCCAGCCACACTTCAGCCTAG-3' and antisense primer, 5'-ACTGTGTGGGTGGGATGTAGCT-3' for MIP-2 (GenBank accession number X53798); sense primer, 5'-ACACGTCTTGCCCTCATTATCTGC-3' and antisense primer, 5'-CCACTCCTTGTTTCTCTCCACG-3' for caspase-1 (GenBank accession number NM_009807); sense primer, 5'-GCGTTGGGTTTTTGTAGATGCC-3' and antisense primer, 5'-ATGTGCTGTCTGATGTCTGGTG-3' for caspase-11 (GenBank accession number Y13089); sense primer, 5'-GAAAGGATGGTTAATGATGCTGAG-3' and antisense primer, 5'-GTCTTCAATGTCCGCATCCTG-3' for BiP (GenBank accession number AJ002387); sense primer, 5'-CATACACCACCACACCTGAAAG-3' and antisense primer, 5'-CCGTTTCCTAGTTCTTCCTTGC-3' for CHOP (GenBank accession number X67083); sense primer, 5'-TCTGATCGGGCGTAGGTTTG-3' and antisense primer, 5'-CATCTCGAAGGGCCTTCTCTAC-3' for Hsc70 (GenBank accession number M19141); sense primer, 5'-TGATGGTGGGAATGGGTCAGAA-3' and antisense primer, 5'-CCAAGAAGGAAGGCTGGAAAAG-3' for beta-actin (GenBank accession number M12481). The primer sets for IL-1beta, MIP-2, caspase-1, caspase-11, BiP, CHOP, Hsc70, and beta-actin are expected to give PCR products with a size of 384, 723, 372, 439, 231, 357, 754, and 674 bp, respectively. PCR consisted of an initial denaturation cycle at 94°C for 2 min, followed by the 27 cycles at 94°C for 15 s, annealing at 55°C for 30 s, and elongation at 68°C for 1 min. An additional cycle at 72°C for 7 min completed the amplification process. Amplified PCR products were separated by 1% agarose gel electrophoresis and visualized with the use of ethidium bromide staining. The primers used for detection of X-box binding protein (XBP) 1 mRNA are as follows: sense primer, 5'-AAACAGAGTAGCAGCGCAGACTGC-3' and antisense primer, 5'-GGATCTCTAAAACTAGAGGCTTGGTG-3'. The primer sets are expected to give PCR products with a size of 600 bp for unspliced form and 574 bp for spliced (active) form XBP1, respectively. PCR consisted of an initial denaturation cycle at 94°C for 2 min, followed by 25 cycles consisting of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and elongation at 68°C for 1 min. An additional cycle at 72°C for 7 min completed the amplification process. The amplified PCR products were separated by 3% agarose gel electrophoresis and then were visualized with ethidium bromide staining.

Immunoblot analysis

The mouse lung was homogenized in lysis buffer (300 mM NaCl, 50 mM Tris-HCl, 1% Triton X-100 (pH 7.5)). After centrifugation, the supernatants served as tissue extracts for an immunoblot analysis. Mouse peritoneal macrophages were homogenized in 20 mM HEPES-KOH (pH 7.5) containing 1% Triton X-100, 20% glycerol, and 1 mM DTT. After centrifugation, the supernatants were used as cell extracts for an immunoblot analysis. In the analysis of CHOP, either lung or peritoneal macrophages were homogenized in 25 mM Tris-HCl (pH 7.4) containing 300 mM NaCl, 1% Triton X-100. After centrifugation, the supernatants served as tissue or cell extracts for an immunoblot analysis. The extracts were then subjected to SDS-PAGE, and then the proteins were electrotransferred to nitrocellulose membranes. Immunodetection was performed using an ECL kit (Amersham Biosciences) according to the protocol provided by the manufacturer.

Measurement of caspase-1 activity

Peritoneal macrophages from wild-type or Chop knockout mice were treated with LPS (150 µg/ml) for 24 h. Caspase-1 activity was measured using the Caspase-1/ICE Colorimetric Assay kit (BioVision) according to the protocol provided by the manufacturer.

Immunohistochemical staining

The lungs were fixed by perfusing them with 4% paraformaldehyde in PBS (pH 7.4), and the excised tissues were immersed in a fixative solution consisting of 4% paraformaldehyde and PBS (pH 7.4) for 3 h at 4°C, washed in PBS for 15 min, dehydrated through a graded series of ethanol and xylene, and embedded in the same paraffin block. Sections (5-µm thick) were cut, air-dried, and deparaffinized. Next, the sections were pretreated with 5 mM periodic acid for 10 min at room temperature to inhibit any endogenous peroxidase activity. The specimens were incubated for 1 h with 100-fold diluted polyclonal Ab against CHOP and/or 100-fold diluted mAb against mouse caspase-11, and then were washed three times with PBS for 5 min. For the single immunostaining of caspase-11, the sections were incubated for 1 h with 500-fold-diluted sheep-anti-mouse Ig (F(ab')2) conjugated with peroxidase (Amersham Biosciences) as a second Ab, and peroxidase activity was visualized by incubation with a 3,3'-diaminobenzidine solution. For the double staining of CHOP and caspase-11, sections were incubated for 1 h with Alexa Fluor 488-labeled anti-rat IgG (Molecular Probes) and Cy3-labeled anti-rabbit IgG as second Abs.

Plasmids and transfection

pcDNA3.1-mCHOP, a mammalian expression plasmid for mouse full-length CHOP, was described (12). The transfection of peritoneal macrophages with plasmids was conducted using Lipofectamine 2000 (Invitrogen Life Technologies) according to the protocol provided by the manufacturer. As a negative control, the same amount of insertless plasmid was transfected.

Statistical analysis

Quantitative results were expressed as the mean ± SD and analyzed using the Student t test. The significance in difference was assigned at a level of less than a 5% probability (p < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LPS-induced lung inflammation was attenuated in Chop knockout mice

LPS was given to wild-type and Chop knockout mice intratracheally. To assess the LPS-induced lung inflammation, a histological analysis with H&E staining was performed (Fig. 1A). In the lung of wild-type mice treated with LPS, severe inflammatory changes, such as thickening of the alveolar septum and the infiltration of many inflammatory cells, were diffusely observed. Such inflammatory changes were obviously attenuated in the lungs of the Chop knockout mice. The infiltration of inflammatory cells in the alveolar space is another specific marker for inflammation in the lung. Therefore, we analyzed the cell types in BALF after LPS treatment (Fig. 1, B–D). Most cells (~90%) in the BALF of the PBS-treated wild-type and Chop knockout mice were macrophages (Fig. 1B). After LPS treatment, the number of cells increased markedly in the wild-type mice (~8 x 105/ml). However, the increase was repressed in the Chop knockout mice (~4 x 105/ml). Next, we counted neutrophils (segmented and stab) and monocytes (including macrophage) after the treatment (Fig. 1D). Segmented neutrophils increased dramatically in the wild-type mice, but only slightly in the Chop knockout mice.


Figure 1
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  		FIGURE 1. The attenuation of LPS-induced inflammation in the lung of Chop knockout mice. A, LPS was given to wild-type and Chop knockout mice intratracheally. After 12 h, the lungs were stained with H & E. Original magnification, x200. B, LPS was given to the wild-type and Chop knockout mice intratracheally. The same volume of PBS was given intratracheally as a negative control. After 12 h, the cells in the BAL fluid were stained with Diff-Quik. Original magnification, x200. C, BAL fluid after LPS treatment was treated the same as in B. The cell numbers in 1 ml of BAL fluid were calculated and the results are shown as means ± SD (n = 4). D, The numbers of segmented neutrophil (Seg), stab neutrophil (Stab), and monocyte including macrophage (Mono) in 1 ml of BAL fluid in C were calculated and the results are shown as the means ± SD (n = 4). E, LPS was given to wild-type and Chop knockout mice intratracheally. After 12 h, the concentration of TNF-{alpha} and IL-1beta in BAL fluid was measured using the ELISA method. The results are shown as means ± SD (n = 5).

 
TNF-{alpha} and IL-1beta are major inflammatory cytokines, which stimulate the synthesis of other inflammatory cytokines, and trigger the start of the inflammatory cascade. Therefore, we measured the concentrations of TNF-{alpha} and IL-1beta in BALF as inflammation markers (Fig. 1E). TNF-{alpha} was barely detected in either the wild-type or Chop knockout mice before LPS treatment. The treatment markedly induced TNF-{alpha} in both the wild-type and Chop knockout mice. There was no difference in the induction level of TNF-{alpha} (~1200 pg/ml) between the wild-type and Chop knockout mice. In contrast, the induction of IL-1beta (active form), which was present at a low level both in nontreated wild-type and Chop knockout mice, was markedly suppressed in the Chop knockout mice. The induced level in LPS-treated wild-type mice was ~150 pg/ml, whereas that in Chop knockout mice was ~50 pg/ml. These results show that LPS-induced lung inflammation is strongly attenuated in the Chop knockout mice, probably due to the suppression of the IL-1beta pathway.

Inflammation of the lung induced by IL-1beta was not attenuated in Chop knockout mice

To investigate whether inflammatory response pathway downstream of IL-1beta is affected in Chop knockout mice, rIL-1beta was given to wild-type and Chop knockout mice intratracheally. To assess the IL-1beta-induced lung inflammation, a histological analysis with H&E staining was performed (Fig. 2A). Severe inflammatory changes, such as thickening of the alveolar septum and the infiltration of many inflammatory cells, were diffusely observed both in the lung of wild-type and Chop knockout mice treated with IL-1beta. Next, we analyzed the cell types in BALF after IL-1beta treatment (Fig. 2, B and C). Most cells (~90%) in the BALF of PBS-treated (0 h) wild-type and Chop knockout mice were monocytes (including macrophages). At 2 h after IL-1beta treatment, the percentage of monocytes was only a little changed both in wild-type and Chop knockout mice BALF. At 6 h after IL-1beta treatment, the percentage of neutrophil was markedly increased (~50%), and the percentage of monocytes was decreased (~45%) both in wild-type and Chop knockout mice BALF. The percentage of lymphocytes was also suppressed at 6 h both in wild-type and Chop knockout mice BALF. These results show that the inflammation cascade downstream of IL-1beta secretion is not affected in Chop knockout mice.


Figure 2
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  		FIGURE 2. The inflammation induced by recombinant mouse IL-1beta (rIL-1beta) in the lung of wild and Chop knockout mice. A, rIL-1beta was given to wild-type and Chop knockout mice intratracheally. The same volume of PBS was given intratracheally as a negative control. After 6 h, the lungs were stained with H & E. Original magnification, x200. B, rIL-1beta was given to wild-type and Chop knockout mice intratracheally. The same volume of PBS was given intratracheally as a negative control (0 h). After the indicated hours, the cells in the BAL fluid were stained with Diff-Quik. Original magnification, x200. C, The percentages of monocyte including macrophage (Mono), neutrophil (Neut), and lymphocyte (Lymp) in 1 ml of BAL fluid in B were calculated and the results are shown as the means ± SD (n = 4).

 
Induction of caspase-11 was suppressed in the LPS-treated lung in Chop knockout mice

IL-1beta is known to act as a proinflammatory cytokine and it also plays a key role in regulating the inflammation cascade (24). To examine whether the activation of the IL-1beta pathway by LPS is suppressed in Chop knockout mice, we analyzed mRNAs for IL-1beta-related proteins in LPS-treated mouse lung by RT-PCR (Fig. 3A). IL-1beta mRNA was not detected before LPS treatment, it was induced almost maximally at 6 h, and thereafter changed only slightly until 24 h in the wild-type mice. In the Chop knockout mouse lung, IL-1beta mRNA was induced by the treatment, but the rate of induction decreased more than in the wild-type mice. mRNA for MIP-2, a chemokine which plays a major role in mediating the chemotaxis of neutrophils in inflammation (25), was not detected before treatment, but it was markedly induced at 6 h, and then it decreased at 24 h, both in wild-type and Chop knockout mice. However, the rate of induction of MIP-2 mRNA decreased in the Chop knockout mice more than in the wild-type mice. The results of IL-1beta and MIP-2 show that LPS treatment triggers the inflammation cascade in the lung of wild-type and Chop knockout mice.


Figure 3
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  		FIGURE 3. Time course of the induction of IL-1beta- and ER stress-related mRNAs and proteins in the LPS-treated mouse lung. A, LPS was given to the wild-type and Chop knockout mice intratracheally. At the indicated times after LPS treatment, total RNAs were isolated from the lungs, and a RT-PCR analysis was performed to detect the indicated mRNAs. beta-actin was used as a control. The data is representative of three independent experiments. B, LPS was given to wild-type and Chop knockout mice intratracheally. At the indicated times after LPS treatment, extracts (25 µg of protein) from lungs were subjected to an immunoblot analysis with Abs against caspase-1 (diluted 1000-fold), caspase-11 (diluted 500-fold), and Hsc70 (diluted 1000-fold). Hsc70 was used as a control. C, The results of caspase-11 in B and in parallel experiments were calculated and are shown as the means ± SD (n = 3). {circ}, Results from the wild-type mice; •, results from the Chop knockout mice. The maximal value is set at 1.0. D, The induction and splicing of XBP1 mRNA in the lung of wild and Chop knockout mice. RT-PCR analysis was performed using total RNAs from the lung at 0 (N) and 6 (L) h after LPS treatment with a primer set encompassing the spliced-out region in XBP1 mRNA. The PCR products were resolved on 3% agarose gels to separate the unspliced (XBP1u) and spliced (XBP1s) XBP1 mRNAs.

 
In stimulated inflammatory cells, IL-1beta is synthesized as pro-IL-1beta, and then it is activated by proteolytic processing before secretion (24), and both caspase-1 and caspase-11 are needed for this processing (19). Caspase-1 mRNA was detected before LPS treatment in the lung of both wild-type and Chop knockout mice, it was slightly induced by LPS treatment in wild-type mice, and it showed almost no change by LPS treatment in Chop knockout mice. In wild-type mice, caspase-11 mRNA was barely detected before treatment and then it was induced markedly at 6 h, and then it decreased at 24 h. In contrast, this induction was strongly attenuated in Chop knockout mice. These results suggest that the attenuation of the induction of IL-1beta (activity) in BALF after LPS treatment in Chop knockout mice (Fig. 1E) is due to the lack of caspase-11 induction. To analyze the induction of caspase-1 and caspase-11 by LPS treatment at the protein level, an immunoblot analysis was performed (Fig. 3, B and C). In the lungs of wild-type mice, caspase-11 was not detected before LPS treatment, while it was induced markedly at 6 h after treatment, and then decreased at 24 h. In sharp contrast, the expression of caspase-11 was not detected in the Chop knockout mice after LPS treatment. In accordance with the results in mRNAs, caspase-1 was detected before treatment, then it was slightly induced at 6 h, and then it remained little changed thereafter in wild-type mice. The induction of caspase-1 was delayed in the Chop knockout mice, and it then gradually increased up to 24 h. The reason for the difference in the time course of caspase-1 induction is unknown. These results show that the induction of caspase-11 by LPS is suppressed in Chop knockout mice. Caspase-11 is needed for the activation of caspase-1 and IL-1beta. Therefore, we conclude that suppression of LPS-induced lung inflammation and the secretion of IL-1beta in BALF in Chop knockout mice is due to the lack of induction of caspase-11.

We also investigated whether the ER stress-CHOP pathway is activated in the LPS-treated mouse lung (Fig. 3A). In wild-type and Chop knockout mice, mRNA for BiP, a major ER chaperone, was detected before treatment, and it was slightly induced by LPS treatment at 6 h, and then it decreased at 24 h. In wild-type mice, CHOP mRNA was barely detected before LPS treatment, then it was induced markedly at 6 h, and thereafter it increased gradually until 24 h. After ER stress, XBP1 mRNA is induced and subjected to unique and specific splicing by activated IRE1, one of the ER stress sensors. Next, the active form of XBP1 is produced, and activates the expression of the ER stress-response genes (26, 27, 28). Therefore, the induction of XBP1 mRNA and the formation of the spliced form of XBP1 mRNA are good markers of the activation of the ER stress pathway. The unspliced form of XBP1 mRNA was expressed at a low level before LPS treatment in wild-type mice (Fig. 3D). It was markedly induced by LPS treatment and its spliced form appeared. Similar results were observed in the Chop knockout mice. This result shows that LPS activates the ER stress pathway in the lung of both wild-type and Chop knockout mice. Therefore, we conclude that LPS treatment induces the ER stress-CHOP pathway in the mouse lung.

Induction of caspase-11 by LPS was suppressed in the peritoneal macrophages from the Chop knockout mice

Regarding inflammation, macrophages play a key role in triggering the inflammation cascade and they are also a major source of IL-1beta secretion. To analyze the direct effect of LPS on macrophages, peritoneal macrophages were prepared from wild-type and Chop knockout mice, and then were treated with LPS (Fig. 4). As shown in Fig. 4A, BiP and caspase-1 mRNAs were moderately induced by LPS in wild-type macrophages, and CHOP, caspase-11, IL-1beta, and MIP-2 mRNAs were markedly induced. In Chop knockout macrophages, the induction of mRNAs for BiP, caspase-1, IL-1beta, and MIP-2 was similar to that of wild-type cells. In contrast, the induction of caspase-11 mRNA was obviously suppressed in the Chop knockout cells. To analyze the induction of caspase-11 and its related molecules at the protein level, immunoblot analysis was performed (Fig. 4B). In wild-type macrophages, neither CHOP nor caspase-11 were detected before LPS treatment, and then they were induced at 24 h after treatment. In contrast, caspase-11 was not induced in the Chop knockout cells. Both in wild-type and Chop knockout cells, caspase-1 was detected before LPS treatment, but it was only slightly induced at 24 h by LPS treatment. BiP was detected at a low level before LPS treatment in both cells, and thereafter it was gradually induced until 12 h in the wild-type cells. In the Chop knockout cells, BiP was only slightly induced at 12 h, and then it decreased at 24 h. CHOP is known to be induced by ER stress. However, CHOP itself was recently reported to activate the formation of reactive oxygen species and abnormal high m.w. protein complexes in ER, and thereafter it enhances ER stress (29). This may be the reason why the induction of BiP mRNA in Chop knockout mice lung by LPS is not as obvious as that seen in wild-type mice. These results show that LPS treatment activates the ER stress-CHOP pathway in macrophages. They also show that the induction of caspase-11 by LPS depends on the CHOP expression in macrophages. As already mentioned, caspase-1 is activated by caspase-11. To examine whether the activation of caspase-1 by LPS treatment is suppressed in Chop knockout macrophages, caspase-1 activity from wild-type or Chop knockout mice macrophages was measured (Fig. 4D). The activity in LPS-treated wild-type macrophages was markedly increased (by about 4-fold) compared with that in nontreated cells. In contrast, the activity in the Chop knockout macrophages was little changed by LPS treatment. These results support the finding that the induction of caspase-11 by LPS is suppressed in peritoneal macrophages from Chop knockout mice.


Figure 4
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  		FIGURE 4. The induction of IL-1beta- and ER stress-related mRNAs and proteins in the LPS-treated mouse peritoneal macrophages. A, Peritoneal macrophages were prepared from wild-type and Chop knockout mice. The cells were treated with LPS (150 µg/ml) for 12 h. A RT-PCR analysis was performed using total RNAs to detect the indicated mRNAs. Hsc70 was used as a control. The data are representative of three independent experiments. B, Peritoneal macrophages were prepared from wild-type and Chop knockout mice. The cells were treated with LPS (150 µg/ml) for the indicated times, and cell extracts (50 µg of protein) were subjected to an immunoblot analysis with Abs against BiP (diluted 500-fold), CHOP (diluted 100-fold), caspase-1 (Casp-1, diluted 1000-fold), caspase-11 (Casp-11, diluted 500-fold), and Hsc70 (diluted 1000-fold). Hsc70 was used as a control. C, The results of caspase-11 in B and in parallel experiments were calculated and are shown as the means ± SD (n = 3). {circ}, Results from wild-type mice; •, results from Chop knockout mice. The maximal value is set at 1.0. D, Peritoneal macrophages from wild-type and Chop knockout mice were treated with LPS (150 µg/ml) for 24 h. Caspase-1 activity was measured as described in Materials and Methods. The activity in nontreated wild-type macrophages is set at 1.0. The results are shown as means ± SD (n = 3).

 
Thapsigargin- and tunicamycin-induced caspase-11 in a CHOP-dependent manner

We next asked whether other ER stress-inducing stimuli induce caspase-11 in macrophages (Fig. 5). Peritoneal macrophages from wild-type and Chop knockout mice were treated with ER stress inducers thapsigargin (Fig. 5A, left panels) and tunicamycin (Fig. 5A, right panels), and mRNAs for ER stress- and IL-1beta-related genes were analyzed by RT-PCR. BiP mRNA was detected before treatment, and it was slightly induced by either thapsigargin or tunicamycin both in wild-type and Chop knockout cells. CHOP mRNA was present at a low level before treatment, and it was markedly induced by either thapsigargin or tunicamycin in wild-type macrophages. These results show the ER stress pathway to thus be activated by thapsigargin and tunicamycin.


Figure 5
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  		FIGURE 5. The induction of caspase-11 by ER stress-inducing reagents in primary cultured mouse macrophages is CHOP dependent. A, Peritoneal macrophages were prepared from wild-type and Chop knockout mice. The cells were treated with thapsigargin (TG, 4 µM) or tunicamycin (TM, 1 µg/ml) for 12 h. A RT-PCR analysis was performed using total RNAs for the detection of the indicated mRNAs. Hsc70 was used as a control. The data are representative of three independent experiments. B, Peritoneal macrophages were prepared from wild-type and Chop knockout mice. The cells were treated with thapsigargin (4 µM) or tunicamycin (1 µg/ml) for the indicated times, and cell extracts (50 µg of protein) were subjected to an immunoblot analysis with Abs against CHOP (diluted 100-fold), caspase-11 (diluted 500-fold), caspase-1 (diluted 1000-fold), BiP (diluted 500-fold), and Hsc70 (diluted 1000-fold). Hsc70 was used as a control. C, The results of CHOP and caspase-11 in B and in parallel experiments were quantified and are shown as the means ± SD (n = 3). {circ}, Results from wild-type cells; •, results from Chop knockout cells. Maximal values are set at 1.0.

 
Caspase-1 mRNA was detected before treatment, and it was slightly changed by these reagents both in wild-type and Chop knockout cells. Caspase-11 was barely detected before treatment, and it was strongly induced by thapsigargin and tunicamycin in wild-type macrophages. In contrast, it was not detected either before and after treatment in Chop knockout cells. IL-1beta and MIP-2 mRNAs were not detected before and after treatment in wild-type and Chop knockout macrophages (data not shown). These results show that the inflammation pathways are not activated by treatment with thapsigargin or tunicamycin. To analyze the induction of caspase-11 and related proteins, an immunoblot analysis was performed (Fig. 5, B and C). When wild type macrophages were treated with thapsigargin or tunicamycin, CHOP which was not detected before treatment, while it was induced at 6 h, reached a maximum at 12 h, and thereafter decreased or remained only slightly changed at 24 h after treatment. In wild type macrophages, caspase-11 was not detected before treatment and then it gradually increased up to 24 h after treatment with thapsigargin or tunicamycin. In Chop knockout cells, caspase-11 was not induced by the treatment with thapsigargin or tunicamycin. Caspase-1 was only slightly changed by the treatment with thapsigargin or tunicamycin both in wild-type and Chop knockout cells. BiP was detected at a low level before treatment, and then it gradually was induced up to 24 h by the treatment with thapsigargin or tunicamycin in both cells. These results show that ER stress induces caspase-11 in macrophages in a CHOP-dependent manner.

CHOP and caspase-11 were coinduced in LPS-treated macrophages

To determine or see whether CHOP and caspase-11 are expressed in the same cells in the lung of LPS-treated mice, an immunohistochemical analysis was performed (Fig. 6). LPS was given to wild-type and Chop knockout mice intratracheally. After 12 h, an immunostaining analysis of the lung with an Ab against caspase-11 was performed (Fig. 6A). Caspase-11-positive cells were not detected in the lung of PBS-treated (control) wild-type mice. We could detect caspase-11-positive cells in the lung of LPS-treated wild-type mice. These cells were morphologically considered to be macrophages. In contrast, caspase-11-positive cells were not detected in the lungs of LPS-treated Chop knockout mice. These results correlate with those of an immunoblot analysis as shown in Fig. 3. The findings of double immunostaining with Abs against CHOP and caspase-11 are shown in Fig. 6B. CHOP and caspase-11 were induced by LPS in the same cells. CHOP was localized in the nuclei, whereas caspase-11 was localized in the cytoplasm. These results show that CHOP and caspase-11 are coinduced in the same cells in the lung of the LPS-treated mice.


Figure 6
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  		FIGURE 6. CHOP and caspase-11 are coinduced in the LPS-treated mouse lung. A, LPS was given to wild-type and Chop knockout mice intratracheally. After 12 h, the lungs were immunostained with an Ab against caspase-11 (diluted 100-fold). The arrows show cells positive for caspase-11. The arrowheads show macrophages negative for caspase-11. Original magnification, x200. B, The lung of a wild-type mouse treated with LPS for 12 h was double-immunostained with Abs against CHOP (diluted 100-fold) and caspase-11 (diluted 100-fold) and then was observed by fluorescence microscopy. CHOP staining, caspase-11 staining, their merged image, and phase-contrast image of the same fields are shown. Original magnification, x200.

 
To examine whether CHOP induces caspase-11, transfection experiments using peritoneal macrophages were performed (Fig. 7A). Peritoneal macrophages from wild-type mice were transfected with an expression plasmid for CHOP, and double immunostaining with Abs against CHOP and caspase-11 was performed. We could thus detect the expression of caspase-11 in CHOP-positive cells. Fig. 7B shows that CHOP and caspase-11 were coinduced in thapsigargin-treated macrophages. In PBS-treated macrophages, CHOP and caspase-11 were barely detected. CHOP and caspase-11 were also coinduced in LPS-treated macrophages (Fig. 7C). These results indicate that LPS induces caspase-11 in macrophages via CHOP induction as ER stress inducers.


Figure 7
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  		FIGURE 7. The expression of CHOP induces caspase-11 in primary cultured peritoneal macrophages. A, Peritoneal macrophages from a wild-type mouse were transfected with CHOP-expression plasmid. At 24 h after transfection, the cells were double-immunostained with Abs against CHOP (diluted 100-fold) and caspase-11 (diluted 100-fold) and then were observed by fluorescence microscopy. CHOP staining, caspase-11 staining, their merged image, and phase-contrast image of the same fields are shown. Original magnification, x200. B, Peritoneal macrophages from a wild-type mouse were treated with thapsigargin (4 µM) for 24 h (upper panels). Treatment with the same volume of PBS was performed as a negative control (lower panels). Next, the cells were double-immunostained with Abs against CHOP (diluted 100-fold) and caspase-11 (diluted 100-fold) and then were observed by fluorescence microscopy. CHOP staining, caspase-11 staining, their merged image, and phase-contrast image of the same fields are shown. Original magnification, x200. C, Peritoneal macrophages from a wild-type mouse were treated with LPS (150 µg/ml) for 24 h (upper panels). Treatment with the same volume of PBS was performed as a negative control (lower panels). Next, the cells were double-immunostained with Abs against CHOP (diluted 100-fold) and caspase-11 (diluted 100-fold) and observed by fluorescence microscopy. CHOP staining, caspase-11 staining, their merged image, and phase-contrast image of the same fields are shown. Original magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Inflammatory stimuli induce apoptosis through various pathways (30). However, little is known about the possible involvement of ER stress- and CHOP-mediated apoptosis in inflammatory diseases and the pathogenesis of lung diseases. Cystic fibrosis is a hereditary disorder of the exocrine glands, and its major symptom is chronic obstructive pulmonary disorder (31). However, the involvement of CHOP in this condition remains unknown. In this report, we found that LPS induces caspase-11 and the activation of IL-1beta through the ER stress-CHOP pathway (Fig. 8). In the early stage of inflammation, the secretion of IL-1beta by macrophages is induced and the following inflammation processes involving the activation of Ag-specific lymphocytes are triggered. Therefore, the induction of caspase-11 and the following activation of caspase-1 are crucial steps in the start of the inflammation cascade. In fact, caspase-1 knockout mice have been reported to be resistant to LPS treatment (17), while caspase-11-deficient oligodendrocytes are resistant to IFN-{gamma}- or TNF-{alpha}-induced cell death (32). Our present work suggests that the ER stress- and the CHOP-mediated pathway plays a key role in inflammation, not only in the lung but also in other tissues, because LPS activates the ER stress pathway in primary cultured peritoneal macrophages. The present study clearly shows that CHOP plays a crucial role in the pathogenesis of LPS-induced inflammation at least in the lung.


Figure 8
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  		FIGURE 8. Summary of the results in this study. Inflammatory stimulation activated the ER stress pathway including CHOP induction. We herein showed that the ER stress-CHOP pathway induces caspase-11. A previous report showed the activation of transcription factor NF-{kappa}B to be involved in the induction of the caspase-11 gene in response to treatment with LPS and IFN-{gamma}. The expression of caspase-11 induces the activation of caspase-1. Next, the complex of activated caspase-1 and caspase-11 increases the activation of pro-IL-1beta to mature IL-1beta. Such activated IL-1beta is therefore considered to trigger the start of the inflammation cascade.

 
Schauvliege et al. (20) reported that caspase-11 induction by LPS and IFN-{gamma} requires NF-{kappa}B and STAT1 in a macrophage cell line. LPS generally induces the activation of NF-{kappa}B and STAT1 in macrophages (33, 34). In the present study, we found that caspase-11 induction by LPS is attenuated in CHOP-deficient macrophages, thus indicating that CHOP is needed for the induction of caspase-11. The activation of p38 MAPK has also been reported to be involved in the induction pathway of caspase-11 (35). CHOP may be involved in p38 MAPK-mediated induction of caspase-11, because the transcriptional enhancer activity of CHOP is enhanced through the phosphorylation by p38 MAPK (36). It remains to be elucidated as to whether CHOP directly activates the transcription of the caspase-11 gene.

The precise mechanism of CHOP-mediated apoptosis is still unknown. CHOP is a transcription factor. Therefore, there must be a target gene(s) which is activated by CHOP and is involved in the apoptosis signal cascade. Wang et al. (37) reported candidate target genes of the CHOP protein by using a representational difference analysis. However, these genes are distinct from the known factors involved in the ER stress response and apoptosis. McCullough et al. (38) reported that CHOP expression results in a down-regulation of the antiapoptotic molecule Bcl-2 expression, the depletion of cellular glutathione, and an exaggerated production of reactive oxygen species. Marciniak et al. (29) reported that CHOP inhibits ER stress-induced attenuation of protein synthesis by the dephosphorylation of the {alpha}-subunit of translation initiation factor 2 (eIF2{alpha}) through the induction of growth arrest and DNA damage-inducible gene 34. They thus showed that the new protein synthesis in ER stress conditions leads to the accumulation of the high m.w. protein complex in ER, and it thus impairs ER function. We showed that the apoptosis signal induced by CHOP is transmitted to the mitochondria through the translocation of a proapoptotic molecule Bax from the cytosol to the mitochondria. However, the mechanism regarding how CHOP induces the translocation of Bax is still unknown. We herein showed that CHOP induces the activation of IL-1beta through the induction of caspase-11 in inflammation. Therefore, we speculate that cytokines, which are induced by IL-1beta, are involved in the induction of apoptosis in inflammatory lesions. Wang et al. (19) reported that the activation of caspase-11 precedes that of caspase-1, caspase-3, and caspase-7. The activation of caspase-3 and caspase-7 was significantly suppressed in the absence of caspase-11. They also showed that the early activation of caspase-3 and caspase-7 by caspase-11 is not affected by blocking caspase-1 activity and IL-1beta release, implying that caspase-11 activates caspase-3 and caspase-7 independently of caspase-1 activation. Caspase-3 and caspase-7 are classified into the apoptosis-effector subgroup of caspases. Therefore, if caspase-3 and caspase-7 are directly activated by caspase-11, then the mitochondria can be skipped in caspase-11-mediated apoptosis. Miura et al. (18) showed that an overexpression of caspase-1 induces apoptosis in cultured cells. They also showed that caspase-1-induced apoptosis is suppressed by Bcl-2. Bcl-2 suppresses apoptosis through the inhibition of the mitochondrial pathway. Therefore, this result suggests that caspase-1-induced apoptosis is mediated by the mitochondrial pathway. We previously reported that Bax is involved in ER stress- and CHOP-mediated apoptosis (18). It remains to be elucidated as to whether Bax is involved in the apoptosis pathway induced by caspase-1 and caspase-11.

The expression of caspase-1 and caspase-11 is not restricted to inflammatory cells (39, 40). Therefore, we speculated that caspase-1 and caspase-11 are also involved in ER stress-mediated apoptosis induced by noninflammatory stimuli. In fact, we found that the ER stress inducers thapsigargin and tunicamycin induce caspase-11 in primary cultured macrophages. The induction of caspase-11 by these ER stress-inducing reagents was CHOP dependent. Caspase-1 is constitutively expressed in macrophages. Therefore, we speculate that caspase-1 and caspase-11 are generally involved in the ER stress- and CHOP-mediated apoptosis pathway, at least in macrophages. The question regarding whether caspase-11 is induced by ER stress in nonmacrophage cells thus remains to be elucidated.

As far as we know, this is the first report showing that the CHOP-caspase-11 pathway plays an important role in the pathogenesis of inflammation in vivo. Severe inflammatory diseases of the lung can be fatal, and the CHOP-caspase-11 pathway may thus become a new target for the therapy of these diseases.


   Acknowledgments
 
We thank our colleagues for their valuable suggestions and discussion, and Brian Quinn for comments on the manuscript. We also thank Rieko Shindo and Yasuko Indo for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants-in-Aid (No. 14037257 to M.M. and No. 16590233 to T.G.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and grants (to T.G.) from the Mitsubishi Pharma Research Foundation and the Inamori Foundation. Back

2 Address correspondence and reprint requests to Dr. Tomomi Gotoh, Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. E-mail address: tomomi{at}gpo.kumamoto-u.ac.jp Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; BiP, Ig H chain binding protein; CHOP, C/EBP homologous protein; Hsc, heat shock cognate protein; BAL, bronchoalveolar lavage; BALF, BAL fluid; XBP, X-box binding protein; ICE, IL-1beta-converting enzyme. Back

Received for publication November 22, 2005. Accepted for publication February 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kleizen, B., I. Braakman. 2004. Protein folding and quality control in the endoplasmic reticulum. Curr. Opin. Cell Biol. 16: 343-349. [Medline]
  2. Schröder, M., R. J. Kaufman. 2005. ER stress and the unfolded protein response. Mutat. Res. 569: 29-63. [Medline]
  3. Blais, J. D., V. Filipenko, M. Bi, H. P. Harding, D. Ron, C. Koumenis, B. G. Wouters, J. C. Bell. 2004. Activating transcription factor 4 is translationally regulated by hypoxic stress. Mol. Cell. Biol. 24: 7469-7482. [Abstract/Free Full Text]
  4. Tajiri, S., S. Oyadomari, S. Yano, M. Morioka, T. Gotoh, J.-I. Hamada, Y. Ushio, M. Mori. 2004. Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ. 11: 403-415. [Medline]
  5. Kouroku, Y., E. Fujita, A. Jimbo, T. Kikuchi, T. Yamagata, M. Y. Momoi, E. Kominami, K. Kuida, K. Sakamaki, S. Yonehara, T. Momoi. 2002. Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation. Hum. Mol. Genet. 11: 1505-1515. [Abstract/Free Full Text]
  6. Rao, R. V., H. M. Ellerby, D. E. Bredesen. 2004. Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 11: 372-380. [Medline]
  7. Oyadomari, S., M. Mori. 2004. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11: 381-389. [Medline]
  8. Gotoh, T., K. Terada, S. Oyadomari, M. Mori. 2004. hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria. Cell Death Differ. 11: 390-402. [Medline]
  9. Oyadomari, S., A. Koizumi, K. Takeda, T. Gotoh, S. Akira, E. Araki, M. Mori. 2002. Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes. J. Clin. Invest. 109: 525-532. [Medline]
  10. Oyadomari, S., K. Takeda, M. Takiguchi, T. Gotoh, M. Matsumoto, I. Wada, S. Akira, E. Araki, M. Mori. 2001. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway. Proc. Natl. Acad. Sci. USA 98: 10845-10850. [Abstract/Free Full Text]
  11. Kawahara, K., S. Oyadomari, T. Gotoh, S. Kohsaka, H. Nakayama, M. Mori. 2001. Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells. FEBS Lett. 506: 135-139. [Medline]
  12. Gotoh, T., S. Oyadomari, K. Mori, M. Mori. 2002. Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J. Biol. Chem. 277: 12343-12350. [Abstract/Free Full Text]
  13. Tsutsumi, S., T. Gotoh, W. Tomisato, S. Mima, T. Hoshino, H. J. Hwang, H. Takenaka, T. Tsuchiya, M. Mori, T. Mizushima. 2004. Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis. Cell Death Differ. 11: 1009-1016. [Medline]
  14. Cavaillon, J. M.. 1994. Cytokines and macrophages. Biomed. Pharmacother. 48: 445-453. [Medline]
  15. Dinarello, C. A.. 1988. Interleukin-1. Ann. NY Acad. Sci. 546: 122-132. [Abstract]
  16. Thornberry, N. A., H. G. Bull, J. R. Calaycay, K. T. Chapman, A. D. Howard, M. J. Kostura, D. K. Miller, S. M. Molineaux, J. R. Weidner, J. Aunins. 1992. A novel heterodimeric cysteine protease is required for interleukin-1beta processing in monocytes. Nature 356: 768-774. [Medline]
  17. Li, P., H. Allen, S. Banerjee, S. Franklin, L. Herzog, C. Johnston, J. McDowell, M. Paskind, L. Rodman, J. Salfeld. 1995. Mice deficient in IL-1beta-converting enzyme are defective in production of mature IL-1beta and resistant to endotoxic shock. Cell 80: 401-411. [Medline]
  18. Miura, M., H. Zhu, R. Rotello, E. A. Hartwieg, J. Yuan. 1993. Induction of apoptosis in fibroblasts by IL-1beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75: 653-660. [Medline]
  19. Wang, S., M. Miura, Y. K. Jung, H. Zhu, E. Li, J. Yuan. 1998. Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92: 501-509. [Medline]
  20. Schauvliege, R., J. Vanrobaeys, P. Schotte, R. Beyaert. 2002. Caspase-11 gene expression in response to lipopolysaccharide and interferon-{gamma} requires nuclear factor-{kappa}B and signal transducer and activator of transcription (STAT) 1. J. Biol. Chem. 277: 41624-41630. [Abstract/Free Full Text]
  21. Salimuddin, R., A. Nagasaki, T. Gotoh, H. Isobe, M. Mori. 1999. Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide. Am. J. Physiol. 277: E110-E117. [Medline]
  22. Schulte, E. K.. 1991. Standardization of biological dyes and stains: pitfalls and possibilities. Histochemistry 95: 319-328. [Medline]
  23. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. [Medline]
  24. Dinarello, C. A.. 1998. Interleukin-1beta, interleukin-18, and the interleukin-1beta converting enzyme. Ann. NY Acad. Sci. 856: 1-11. [Abstract/Free Full Text]
  25. Driscoll, K. E.. 1994. Macrophage inflammatory proteins: biology and role in pulmonary inflammation. Exp. Lung Res. 20: 473-490. [Medline]
  26. Yoshida, H., T. Matsui, A. Yamamoto, T. Okada, K. Mori. 2001. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107: 881-891. [Medline]
  27. Lee, K., W. Tirasophon, X. Shen, M. Michalak, R. Prywes, T. Okada, H. Yoshida, K. Mori, R. J. Kaufman. 2002. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev. 16: 452-466. [Abstract/Free Full Text]
  28. Calfon, M., H. Zeng, F. Urano, J. H. Till, S. R. Hubbard, H. P. Harding, S. G. Clark, D. Ron. 2002. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415: 92-96. [Medline]
  29. Marciniak, S. J., C. Y. Yun, S. Oyadomari, I. Novoa, Y. Zhang, R. Jungreis, K. Nagata, H. P. Harding, D. Ron. 2004. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev. 18: 3066-3077. [Abstract/Free Full Text]
  30. Martinon, F., J. Tschopp. 2004. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell 117: 561-574. [Medline]
  31. Okiyoneda, T., K. Harada, M. Takeya, K. Yamahira, I. Wada, T. Shuto, M. A. Suico, Y. Hashimoto, H. Kai. 2003. {Delta}F508 CFTR pool in the ER is increased by calnexin overexpression. Mol. Biol. Cell 15: 563-574. [Medline]
  32. Hisahara, S., J. Yuan, T. Momoi, H. Okano, M. Miura. 2001. Caspase-11 mediates oligodendrocyte cell death and pathogenesis of autoimmune-mediated demyelination. J. Exp. Med. 193: 111-122. [Abstract/Free Full Text]
  33. Müller, J. M., H. W. Ziegler-Heitbrock, P. A. Baeuerle. 1993. Nuclear factor {kappa}B, a mediator of lipopolysaccharide effects. Immunobiology 187: 233-256. [Medline]
  34. Ohmori, Y., T. A. Hamilton. 2001. Requirement for STAT1 in LPS-induced gene expression in macrophages. J. Leukocyte Biol. 69: 598-604. [Abstract/Free Full Text]
  35. Hur, J., S. Y. Kim, H. Kim, S. Cha, M. S. Lee, K. Suk. 2001. Induction of caspase-11 by inflammatory stimuli in rat astrocytes: lipopolysaccharide induction through p38 mitogen-activated protein kinase pathway. FEBS Lett. 507: 157-162. [Medline]
  36. Wang, X. Z., D. Ron. 1996. Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP kinase. Science 272: 1347-1349. [Abstract]
  37. Wang, X. Z., M. Kuroda, J. Sok, N. Batchvarova, R. Kimmel, P. Chung, H. Zinszner, D. Ron. 1998. Identification of novel stress-induced genes downstream of chop. EMBO J. 17: 3619-3630. [Medline]
  38. McCullough, K. D., J. L. Martindale, L. O. Klotz, T. Y. Aw, N. J. Holbrook. 2001. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol. Cell. Biol. 21: 1249-1259. [Abstract/Free Full Text]
  39. Li, M., V. O. Ona, C. Guegan, M. Chen, V. Jackson-Lewis, L. J. Andrews, A. J. Olszewski, P. E. Stieg, J. P. Lee, S. Przedborski, R. M. Friedlander. 2000. Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model. Science 288: 335-339. [Abstract/Free Full Text]
  40. Kang, S. J., S. Wang, H. Hara, E. P. Peterson, S. Namura, S. Amin-Hanjani, Z. Huang, A. Srinivasan, K. J. Tomaselli, N. A. Thornberry, et al 2000. Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions. J. Cell Biol. 149: 613-622. [Abstract/Free Full Text]

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